JP6536411B2 - Medium composition - Google Patents

Description

  The present invention relates to a medium composition for culturing animal and plant cells and / or tissues, particularly in three dimensions or in suspension, using nanofibers such as polysaccharides having enhanced dispersibility in water, and uses thereof .

  In recent years, techniques for growing or maintaining various organs, tissues and cells playing different roles in animals and plants in vitro have been developed. Proliferating or maintaining these organs and tissues in vitro is called organ culture and tissue culture, respectively, and proliferating, differentiating or maintaining cells isolated from organs and tissues in vitro It is called cell culture. Cell culture is a technology to proliferate, differentiate or maintain isolated cells in vitro in a culture medium, and is essential for detailed analysis of the functions and structures of various organs, tissues and cells in vivo. ing. In addition, cells and / or tissues cultured by this technology can be evaluated for efficacy and toxicity of chemical substances and drugs, mass production of useful substances such as enzymes, cell growth factors and antibodies, and organs lost due to diseases or defects. It is used in a variety of fields such as tissue and tissue regenerative medicine, plant breed improvement, and production of genetically modified crops.

  Because of their properties, animal-derived cells are roughly divided into floating cells and adherent cells. Floating cells are cells that do not require a scaffold for growth and proliferation, and adherent cells are cells that require a scaffold for growth and proliferation, but most of the cells that make up the organism are the latter adherent cells. . As culture methods for adherent cells, monolayer culture, dispersion culture, embedding culture, microcarrier culture, sphere culture and the like are known.

  Monolayer culture is a culture vessel consisting of glass or synthetic polymer material subjected to various surface treatments, and a method of culturing target cells in a monolayer form by using auxiliary cells called feeder cells as a scaffold. Are widely used. For example, various types of surface treatment (plasma treatment, corona treatment, etc.) to polystyrene, those coated with cell adhesion factor such as collagen, fibronectin, polylysine, or those previously seeded with feeder cells, etc. Culture methods have been developed using culture vessels of the shape or nature. However, monolayer culture can not maintain the specific function of cells in vivo for a long time because the two-dimensional culture environment is completely different from the environment in vivo. The problem is that it can not reconstruct such tissues, and is not suitable for mass culture of cells because the number of cells per a certain area is limited (Patent Document 1). In addition, in the method of culturing target cells on feeder cells, separation of the feeder cells and the target cells may be problematic (Non-patent Document 1).

  In the dispersion culture, after the cells are seeded in the culture medium, adhesion of the cells to the culture vessel is inhibited by continuing the agitation of the culture solution in a culture vessel subjected to a surface treatment to inhibit the adhesion of the cells. It is a method of culturing adherent cells in a suspended state. However, adherent cells cultured by this method can not adhere to the scaffold, and therefore, they can not be applied to cells that require adhesion to the scaffold for cell growth. Moreover, since it can not exhibit an original cell function by being always crushed by shear force, it may be unable to culture a cell which has a function in large quantities (nonpatent literature 2).

  The embedded culture is a method of embedding and immobilizing cells in a solid or semisolid gel substrate such as agar, methyl cellulose, collagen gel, gelatin, fibrin, agarose, alginate, etc., and culturing. The method makes it possible to culture cells three-dimensionally in a form close to in vivo, and since the gel substrate itself may promote cell growth and differentiation, it may be compared with monolayer culture or dispersion culture. It is possible to culture cells at high density while maintaining the function of the cells (Patent Documents 2 and 3). Furthermore, a method has also been developed in which microcapsules having a size of 100 to 300 μm are prepared in a state in which the cells are embedded in these gel substrates, and the cells are cultured in an aqueous medium while dispersing the microcapsules (Non-patent literature 3). However, these methods do not allow time-lapse observation of cultured cells when the gel substrate does not transmit visible light. The medium and microcapsules containing the gel substrate have high viscosity, so the cells are recovered from the medium Have problems such as enzyme treatment (for example, collagenase treatment in the case of collagen gel) which requires complicated operations such as collagenase treatment and damage to the cells, and it is difficult to replace the medium required for long-term culture. doing. In recent years, technology has been developed that enables recovery of cells from a gel substrate by treatment such as heat and shear force, but heat and shear force may adversely affect cell function, and the gel substrate The safety of the compound for the living body has not been clarified yet (patent documents 4, 5, non-patent documents 4, 5, 6, 7). In addition, sol-like food products have been developed in the food field to uniformly disperse and suspend small-cut granular foods such as fruits and vegetables and prevent their precipitation and floating, but the sol-like food was dispersed. It is not considered to recover granular food, and it has not been examined whether cells and tissues can be cultured in suspension (Patent Document 6). It is known that gellan in an aqueous solution gels by the action of calcium ions to form a fine structure (Non-patent Document 8).

In microcarrier culture, cells are grown in a monolayer on the surface of microparticles (hereinafter also referred to as microcarriers) slightly heavier than water, and the microparticles are agitated in a culture vessel such as a flask and cultured in a suspended state To do. Usually, the microcarriers used in the method are spherical particles having a diameter of 100 to 300 μm, a surface area of 300 to 6000 cm ² / g, and a specific gravity of 1.03 to 1.05, and are made of materials such as dextran, gelatin, alginic acid or polystyrene. ing. A charged group such as collagen, gelatin or dimethylaminoethyl can also be provided on the surface of the microcarrier to facilitate cell attachment. The method is applied to mass culture of cells because it enables the culture area to be extremely increased (Patent Documents 7 and 8). However, it is difficult to attach the target cells almost uniformly to all the microcarriers, and the shear force during stirring causes the cells to detach from the microcarriers, causing the cells to be damaged, etc. (Non-Patent Document 9).

  Sphere culture is a method in which cells of interest form tens or hundreds of aggregates (hereinafter also referred to as spheres), and then the aggregates are cultured by standing or shaking in a culture medium. is there. Spheres have high cell density, cell-cell interactions and cell structures close to their in vivo environment, and can be cultured while maintaining cell function longer than monolayer culture and dispersion culture methods. Are known (non-patent documents 10 and 11). However, sphere culture can not form large spheres because it is difficult to supply nutrients and excrete waste inside the sphere if the size of the sphere is too large. In addition, since it is necessary to culture the formed spheres in a dispersed state at the bottom of the culture vessel, it is difficult to increase the number of spheres per constant volume, which is not suitable for mass culture. Furthermore, as a method for producing the sphere, hanging drop culture, culture on a cell non-adherent surface, culture in a microwell, spin culture, culture using a cell scaffold, aggregation by centrifugal force, ultrasonic wave, electric field or magnetic field Although these methods are known, their operation is complicated, recovery of spheres is difficult, size control and mass production are difficult, effects on cells are unknown, special special containers and devices are required, etc. (Patent Document 9).

  On the other hand, regarding plants, cells, protoplasts from which cell walls have been removed, leaves, stems, roots, growing points, organs such as seeds, embryos, pollen, tissues, and callus can be cultured and grown in a sterile state. Using such plant tissue and cell culture techniques, plant variety improvement and useful substance production are also possible. A method of suspending and culturing plant cells and tissues in a liquid medium is known as a method for growing plant cells and tissues in a large amount in a short period of time (Non-patent Document 12). In order to achieve their good growth, it is important to supply a sufficient amount of oxygen, maintain a uniform mixing state, and further to prevent cell breakage. The supply of oxygen to the culture solution and the suspension of cells and tissues may be performed by combining aeration and mechanical agitation, or may be performed only by aeration. Although breakage may lead to poor growth due to breakage, while the latter may cause less shear of cells and tissues, it may be difficult to maintain a uniform mixed state in high density culture, so cells and tissues There are problems such as sedimentation and reduction of proliferation efficiency.

JP 2001-128660 A JP-A-62-171680 JP-A-63-209581 JP, 2009-29967, A Japanese Patent Application Publication No. 2005-60570 Unexamined-Japanese-Patent No. 8-23893 JP 2004-236553 A International Publication No. 2010/059775 JP, 2012-65555, A

Klimanskaya et al., Lancet 2005, 365: 1636-1641. King et al., Curr Opin Chem Biol. 2007, 11: 394-398. Murua et al. of Controlled Release 2008, 132: 76-83. Mendes, Chemical Society Reviews 2008, 37: 2512-2529 Moon et al., Chemical Society Reviews 2012, 41: 4860-4883 Pek et al., Nature Nanotechnol. 2008, 3: 671-675 Liu et al., Soft Matter 2011, 7: 5430-5436. Perez-Campos et al., Food Hydrocolloids 2012, 28: 291-300 Leung et al., Tissue Engineering 2011, 17: 165-172. Stahl et al., Biochem. Biophys. Res. Comm. 2004, 322: 684-692 Lin et al., Biotechnol J. 2008, 3: 1172-1184 Weathers et al., Appl Microbiol Biotechnol 2010, 85: 1339-1351

  The object of the present invention is to solve the above-mentioned problems of the prior art, and to use a culture medium composition for culturing animal and plant cells and / or tissues, particularly in three dimensions or in suspension, and plant and animal cells using the culture medium composition And / or providing a method of culturing the tissue.

  As a result of intensive studies, the inventors of the present invention have found that animal and plant cells and / or tissues can be obtained without substantially increasing the viscosity of the liquid medium by mixing nanofibers made of polysaccharides such as cellulose and chitin into the liquid medium. It has been found that suspension culture can be performed in the state of standing still, and the proliferation activity of the cells can be enhanced by culturing using this medium composition. Moreover, not only water-insoluble polysaccharides such as cellulose but also water-soluble polysaccharides such as deacylated gellan gum form a fibrous structure in the liquid medium, which substantially increases the viscosity of the liquid medium. It has been found that suspension culture of animal and plant cells and / or tissues can be performed without raising them. Furthermore, it has also been found that cultured cells and / or tissues can be easily recovered from these medium compositions. Based on the above findings, the present invention has been completed by further investigation.

  That is, the present invention is as follows:

[1] A medium composition capable of suspending and culturing cells or tissues, which comprises nanofibers.
[2] The medium composition according to [1], wherein cells or tissues can be recovered after the exchange process of the medium composition at the time of culture and the completion of culture.
[3] The medium composition of [1], which does not require any change in temperature, chemical treatment, enzyme treatment, or shear force when recovering cells or tissues.
[4] The medium composition of [1], which has a viscosity of 8 mPa · s or less.
[5] The nanofibers are characterized in that the average fiber diameter of the nanofibers is 0.001 to 1.00 μm, and the ratio (L / D) of the average fiber length (L) to the average fiber diameter (D) is 2 to 500. Medium composition of [1].
[6] The medium composition of [1], wherein the nanofibers are composed of a polymer compound.
[7] The medium composition of [6], wherein the polymer compound is a polysaccharide.
[8] The polysaccharide is
Any water-insoluble polysaccharide selected from the group consisting of cellulose, chitin and chitosan; or hyaluronic acid, gellan gum, deacylated gellan gum, rhamsan gum, diutatan gum, xanthan gum, carrageenan, xanthan gum, hexuronic acid, fucoidan, pectin A water-soluble polysaccharide selected from the group consisting of pectic acid, pectinic acid, heparan sulfate, heparin, heparitin sulfate, kerato sulfate, chondroitin sulfate, dermatan sulfate, rhamnan sulfate, alginic acid and salts thereof, [7] Medium composition.
[9] The medium composition of [8], wherein the polysaccharide comprises cellulose or chitin.
[10] The medium composition according to [9], wherein the nanofibers are obtained by grinding.
[11] The medium composition according to any one of [1] to [10], which is for cell culture.
[12] The medium composition of [11], wherein the cells are adherent cells or floating cells.
[13] The medium composition of [12], wherein the adherent cells are spheres.
[14] A cell or tissue culture comprising the medium composition according to any one of [1] to [13] and a cell or tissue.
[15] A method for culturing cells or tissues, comprising culturing the cells or tissues in the medium composition described in any one of [1] to [13].
[16] A method for recovering cells or tissues, comprising separating cells or tissues from the culture of [14].
[17] The recovery method of [16], wherein the separation is performed by centrifugation.
[18] A method for producing a sphere comprising culturing adherent cells in the medium composition of any of [1] to [13].
[19] A medium additive for preparing a medium composition according to any one of [1] to [13], which comprises the nanofiber or the water-soluble polymer compound constituting the nanofiber. Medium additives.
[20] A method for producing a medium composition, which comprises mixing a medium additive and a medium according to [19].
[21] A method for producing the medium composition according to any one of [1] to [13], which comprises mixing the nanofibers or the water-soluble polymer compound constituting the nanofibers with the culture medium Method of making the composition.
[22] A method for preserving cells or tissues, which comprises preserving cells or tissues in the medium composition described in any of [1] to [13].
[23] A method of transporting cells or tissues, which transports cells or tissues in the medium composition of any of [1] to [13].
[24] A method for growing cells or tissues, comprising culturing the cells or tissues in the medium composition described in any one of [1] to [13].
[25] A method for subculturing adherent cells, comprising the following steps:
(1) Suspension-adhering adherent cells in the medium composition according to any one of [1] to [13]; and (2) step (i) without removing cells from the culture vessel Freshly, the culture medium composition according to any one of [1] to [13] is added to a culture containing adherent cells obtained by suspension culture of (1), or (ii) fresh [1] Adding all or part of a culture containing adherent cells obtained by the suspension culture of step (1) to the medium composition described in any of [13].
[26] A method for growing adherent cells, comprising suspending and culturing adherent cells in a state of being attached to the chitin nanofibers in a medium composition containing chitin nanofibers.
[27] The method according to [26], wherein the content of chitin nanofibers in the medium composition is 0.0001% (w / v) or more and 0.1% (w / v) or less.

  The present invention provides a media composition comprising nanofibers, in particular comprised of polysaccharides. When the medium composition is used, cells and / or tissues can be cultured in a floating state without operations such as shaking or rotation which may cause damage or loss of function of cells or tissues. Furthermore, using the medium composition, the medium can be easily exchanged during culture, and the cultured cells and / or tissues can be easily recovered. The culture method can be applied to cells and / or tissues collected from animal organisms or plants, and target cells and / or tissues can be prepared in large amounts without impairing their functions. And cells and / or tissues obtained by the culture method are evaluated for efficacy and toxicity of chemicals, pharmaceuticals and the like, mass production of useful substances such as enzymes, cell growth factors and antibodies, and organs lost due to diseases or defects. , And tissue and cells can be used when performing regenerative medicine and the like.

  In addition, since the medium composition of the present invention can be used to maintain cells or tissues in an environment close to in vivo, it is useful for preservation and transport of cells and tissues. For example, when adherent culture of cells on a plate and transporting it as it is, the vibration during transport may cause the cells to detach from the plate, etc., resulting in a decrease in the original function of the cells. In the medium composition of the present invention, the nanofibers form a three-dimensional network, which can support the cells, thereby holding the cells in a suspended state, and therefore cell damage due to detachment from the plate due to vibration during transport. It can be avoided and cells can be stored and transported while maintaining their original function.

When the spheres of HepG2 cells are cultured with the medium composition, the spheres are uniformly dispersed and can be cultured in suspension. When the spheres of HeLa cells are cultured with the medium composition, the spheres are uniformly dispersed and can be cultured in a floating state. When the spheres of HeLa cells are cultured in the medium composition and the spheres are observed microscopically, it is a figure showing that association of the spheres is suppressed as compared to the existing medium. When the microcarriers to which HepG2 cells were attached with a culture medium composition are cultured, it is a figure which shows that HepG2 cells can be proliferated on a microcarrier. When HeLa cell spheres are added to the medium composition, the spheres are uniformly dispersed and in suspension. It is a figure which shows that the medium composition forms the sphere of HeLa cells. It is a figure which shows the film which is one aspect | mode of a structure. The concentration of deacylated gellan gum to the media composition is 0.02% (w / v). It is a figure which shows that the medium composition forms the sphere of HepG2 cells. It is a figure which shows the floating state at the time of culture | cultivating a laminin coat GEM which made HepG2 cell adhere on a culture medium composition. It is a figure which shows the floating state at the time of culturing the alginate bead which embedded HepG2 cell with a culture medium composition. It is a figure which shows the floating state at the time of culturing the collagen gel capsule which embedded HepG2 cell by culture-medium composition. It is a figure which shows the floating state at the time of culture | cultivating a rice origin callus with a culture medium composition. The viscosity of each culture medium composition at 25 ° C. is shown. Fig. 2 shows a scanning electron micrograph of the MNC-containing medium composition of Example 1. FIG. 6 shows a scanning electron micrograph of the PNC-containing medium composition of Example 2. FIG. 17 shows a scanning electron micrograph of the CT-containing medium composition of Example 3. FIG. 16 shows a scanning electron micrograph of the DAG-containing medium composition of Example 4. FIG. 17 shows a scanning electron micrograph of the Car-containing medium composition of Example 5. Dry at room temperature. 17 shows a scanning electron micrograph of the Car-containing medium composition of Example 5. Dry at 110 ° C. 17 shows a scanning electron micrograph of the Xan-containing medium composition of Comparative Example 3. 17 shows a scanning electron micrograph of the DU-containing medium composition of Comparative Example 4. The results of observation of the dispersed state of spheres after suspension culture of the spheres of HepG2 cells for 6 days in the MNC-containing medium composition of Example 1 are shown. From the left, the MNC concentration is 0.01, 0.03, 0.05, 0.07 and 0.1 w / v%. The results of observation of the dispersed state of the spheres after suspension culture of the spheres of HepG2 cells for 6 days in the PNC-containing medium composition of Example 2 are shown. From the left, PNC concentrations are 0.01, 0.03, 0.05, 0.07 and 0.1 w / v%. The observation results of the dispersed state of the spheres after suspension culture of the spheres of HepG2 cells for 6 days in the CT-containing medium composition of Example 3 are shown. From the left, CT concentrations are 0.01, 0.03, 0.05, 0.07 and 0.1 w / v%. The results of observation of the dispersed state of the spheres after suspension culture of the spheres of HepG2 cells for 6 days in the DAG-containing medium composition of Example 4 are shown. From the left, DAG concentrations are 0.01, 0.03, 0.05, 0.07 and 0.1 w / v%. The results of observation of the dispersed state of the spheres after suspension culture of the spheres of HepG2 cells for 6 days in the Car-containing medium composition of Example 5 are shown. From the left, the Car concentration is 0.01, 0.03, 0.05, 0.07 and 0.1 w / v%. The observation results of the dispersed state of the spheres after suspension culture of the spheres of HepG2 cells for 6 days in the Xan-containing medium composition of Example 5 are shown. From the left, the Xan concentrations are 0.01, 0.03, 0.05, 0.07 and 0.1 w / v%. The observation results of the dispersed state of the spheres after suspension culture of the spheres of HepG2 cells for 6 days in the DU-containing medium composition of Comparative Example 4 are shown. From the left, DU concentrations are 0.01, 0.03, 0.05, 0.07 and 0.1 w / v%. The observation results of the dispersed state of the spheres after suspension culture of the spheres of HepG2 cells for 6 days in the Alg-containing medium composition of Comparative Example 5 are shown. From the left, the Alg concentrations are 0.01, 0.03, 0.05, 0.07 and 0.1 w / v%. It shows RLU values at day 6 after suspension culture of MCF7 cells was started in the medium compositions of Example 1 'and Comparative Example 5'. The MCF7 cells show RLU values on day 6 after suspension culture was initiated in the medium compositions of Examples 2 'and 3'. The MCF7 cells show RLU values on day 6 after suspension culture was initiated in the medium compositions of Example 4 'and Example 5'. The MCF7 cells show RLU values on day 6 after suspension culture was initiated in the medium compositions of Comparative Examples 3 'and 4'. Fig. 16 shows the RLU values at day 6 after suspension culture of A375 cells was started in the medium compositions of Example 1 'and Comparative Example 5'. The R375 values are shown on day 6 after suspension culture of A375 cells was initiated in the medium compositions of Examples 2 'and 3'. The R375 values are shown on day 6 after suspension culture of A375 cells was started in the medium compositions of Example 4 'and Example 5'. The R375 values are shown on day 6 after suspension culture of A375 cells was initiated in the medium compositions of Comparative Examples 3 'and 4'. It is the result of observing microscopically the dispersed state of MCF7 cells in each medium composition on the second day of suspension culture start. It is the result of observing microscopically the dispersion state of A375 cells in each culture medium composition on the second day of suspension culture start. It is the result of carrying out the microscopic observation of the dispersed state of the MDCK cell in each culture medium composition in the 4th day of suspension culture start.

Hereinafter, the present invention will be described in more detail.
The terms used in the present specification are defined as follows.

  The cell in the present invention is the most basic unit constituting an animal or a plant, and has as its element the cytoplasm and various organelles inside the cell membrane. At this time, the nucleus that encapsulates the DNA may or may not be contained inside the cell. For example, cells derived from animals in the present invention include germ cells such as sperm and egg, somatic cells that constitute a living body, stem cells, precursor cells, cancer cells separated from living bodies, and cells obtained from living bodies to obtain immortalization ability. And cells which are stably maintained outside the body (cell lines), cells which have been separated from living organisms and have been artificially modified genetically, and cells which have been separated from living organisms and which have artificially exchanged nuclei are included. Examples of somatic cells that constitute a living body include, but are not limited to, fibroblasts, bone marrow cells, B lymphocytes, T lymphocytes, neutrophils, red blood cells, platelets, macrophages, monocytes, bones Cells, bone marrow cells, pericytes, dendritic cells, keratinocytes, adipocytes, mesenchymal cells, epithelial cells, epidermal cells, endothelial cells, endothelial cells, hepatic parenchymal cells, chondrocytes, cumulus cells, neural cells, Glial cells, neurons, oligodendrocytes, microglia, astrocytes, cardiac cells, esophageal cells, muscle cells (eg, smooth muscle cells or skeletal muscle cells), pancreatic beta cells, melanocytes, hematopoietic progenitor cells, and single cells Nuclear cells etc. are included. The somatic cells include, for example, skin, kidney, spleen, adrenal, liver, lung, ovary, pancreas, uterus, stomach, colon, small intestine, large intestine, large intestine, spleen, bladder, prostate, testis, thymus, muscle, connective tissue, bone, cartilage And cells collected from any tissue such as blood vessel tissue, blood, heart, eye, brain or nerve tissue. A stem cell is a cell having both the ability to replicate itself and the ability to differentiate into cells of multiple other lineages, examples of which include, but are not limited to, embryonic stem cells (ES cells) , Embryonic tumor cells, embryonic germ stem cells, induced pluripotent stem cells (iPS cells), neural stem cells, hematopoietic stem cells, mesenchymal stem cells, hepatic stem cells, pancreatic stem cells, muscle stem cells, germ stem cells, intestinal stem cells, cancer stem cells, It includes hair follicle stem cells and the like. The precursor cells are cells in the process of differentiating from the stem cells to specific somatic cells or germ cells. Cancer cells are cells derived from somatic cells and having acquired infinite proliferation ability. A cell line is a cell that has acquired infinite proliferation ability by artificial manipulation in vitro, and examples thereof include, but are not limited to, CHO (Chinese hamster ovary cell line), HCT116 , Huh7, HEK293 (human embryonic kidney cells), HeLa (human uterine cancer cell line), HepG2 (human liver cancer cell line), UT7 / TPO (human leukemia cell line), MDCK, MDBK, BHK, C-33A, HT- 29, AE-1, 3D9, Ns0 / 1, Jurkat, NIH 3T3, PC12, S2, Sf9, Sf21, High Five (registered trademark), Vero and the like.

  The plant-derived cells in the present invention include cells isolated from each tissue of plants, and also include protoplasts obtained by artificially removing cell walls from the cells.

  The tissue in the present invention is a unit of a structure in which cells having several different properties and functions are assembled in a certain manner, and examples of animal tissues include epithelial tissue, connective tissue, muscle tissue, nerve. Organization etc. are included. Examples of plant tissue include meristematic tissue, epidermal tissue, anabolic tissue, mesophyll tissue, passage tissue, mechanical tissue, parenchyma, dedifferentiated cell mass (callus) and the like.

  When culturing cells and / or tissues, cells and / or tissues to be cultured can be arbitrarily selected and cultured from the cells and / or tissues described above. Cells and / or tissues can be harvested directly from animals or plants. Cells and / or tissues may be harvested after being induced, grown or transformed from animals or plants by specific treatment. At this time, the process may be in vivo or in vitro. Examples of the animal include fish, amphibians, reptiles, birds, pan crustaceans, hexapods, mammals and the like. Examples of mammals include, but are not limited to, rats, mice, rabbits, guinea pigs, squirrels, hamsters, voles, platypus, dolphins, whales, dogs, cats, goats, cattle, horses, sheep, pigs, elephants , Common marmosets, squirrel monkeys, rhesus monkeys, chimpanzees and humans. The plant is not particularly limited as long as the collected cells and / or tissues can be liquid-cultured. For example, plants that produce herbal medicines (eg, saponins, alkaloids, berberine, scopolin, plant sterols, etc.) (eg, ginseng, periwinkle, hyos, hyacinth, ouren, veradonna etc.), pigments and polysaccharides that become cosmetics and food materials Plants that produce the body (for example, anthocyanin, safflower pigment, raspberry pigment, saffron pigment, flavones, etc.) (for example, blueberry, safflower, red radish, saffron etc), or plants that produce the drug substance Not limited to them.

  In the present invention, suspension of cells and / or tissues means that cells and / or tissues do not adhere to the culture vessel (non-adhesion). Furthermore, in the present invention, when proliferating, differentiating or maintaining cells and / or tissues, the cells and / or tissues are not subjected to external pressure or vibration to the liquid medium composition or shaking or rotation in the composition. A state in which the tissue is uniformly dispersed in the liquid medium composition and is in a floating state is referred to as "floating and standing", and culturing cells and / or tissues in the state is referred to as "floating and stationary culture". It is said. The period in which "floating and standing" can be suspended is at least 5 minutes or more, preferably 1 hour or more, 24 hours or more, 48 hours or more, 6 days or more, 21 days or more, but the floating state is maintained. As long as it is not limited to these periods.

  In a preferred embodiment, the medium composition of the present invention is capable of suspending cells and / or tissues at least at one point of a temperature range (for example, 0 to 40 ° C.) at which cells and tissues can be maintained and cultured. is there. The medium composition of the present invention is capable of suspending cells and / or tissues at preferably at least one point in the temperature range of 25 to 37 ° C, most preferably at 37 ° C.

  For example, polystyrene beads (Size 500-600 μm, manufactured by Polysciences Inc.) are uniformly dispersed in the medium composition to be evaluated, and allowed to stand at 25 ° C. It can be evaluated by observing whether or not the suspended state of the cells is maintained for minutes or more (preferably, 24 hours or more, 48 hours or more).

The medium composition of the present invention is a composition containing nanofibers and a medium in which cells or tissues can be suspended and cultured (preferably suspended and cultured).
The medium composition is preferably a composition capable of recovering cells or tissues after the exchange process of the medium composition at the time of culture and completion of culture, and more preferably, at the time of cell or tissue recovery, It is a composition that does not require any temperature change, chemical treatment, enzyme treatment, or shear force.

[Nanofiber]
The nanofibers contained in the medium composition of the present invention show the effect of uniformly suspending cells and / or tissues in a liquid medium. More specifically, low molecular weight compounds and high molecular weight compounds assemble and self-assemble through covalent bonds, ionic bonds, electrostatic interactions, hydrophobic interactions, van der Waals forces, etc. to form nanofibers in a liquid medium. Alternatively, nanofibers obtained by micronizing a relatively large fiber structure made of a polymer compound by high-pressure treatment or the like may be mentioned as the nanofibers contained in the medium composition of the present invention. Although not being bound by theory, in the medium composition of the present invention, the nanofibers form a three-dimensional network, which supports the cells and tissues, thereby maintaining the cells and tissues suspended.

  In the present specification, nanofibers refer to fibers having an average fiber diameter (D) of 0.001 to 1.00 μm. The average fiber diameter of the nanofibers used in the present invention is preferably 0.005 to 0.50 μm, more preferably 0.01 to 0.05 μm, and still more preferably 0.01 to 0.02 μm. If the average fiber diameter is less than 0.001 μm, there is a risk that the floating effect may not be obtained due to the nanofiber being too fine, which may not lead to the improvement of the characteristics of the medium composition containing it.

  The aspect ratio (L / D) of the nanofibers used in the present invention is obtained from the average fiber length / average fiber diameter, and is usually 2 to 500, preferably 5 to 300, and more preferably 10 to 250. . If the aspect ratio is less than 2, the dispersibility in the medium composition is lacking, and there is a possibility that the floating action can not be sufficiently obtained. If it exceeds 500, it means that the fiber length becomes extremely large, and there is a possibility that the passage operation such as medium exchange may be disturbed by increasing the viscosity of the composition. In addition, the composition of the medium is less likely to transmit visible light, which leads to a decrease in transparency, which makes it difficult to observe cultured cells over time, and it interferes with cell evaluation using light absorption, fluorescence, luminescence, etc. there is a possibility.

  In the present specification, the average fiber diameter (D) of nanofibers was determined as follows. First, hydrophilize the collodion support membrane from Soken Shoji Co., Ltd. using ion cleaner (JIC-410) from Nippon Denshi Co., Ltd. for 3 minutes, and measure the number of nanofiber dispersions to be evaluated (diluted with ultra pure water) It was added dropwise and dried at room temperature. This is observed with a transmission electron microscope (TEM, H-8000) (10,000 times) manufactured by Hitachi, Ltd. at an acceleration voltage of 200 kV, and using the obtained image, the number of samples: 200 to 250 The fiber diameter of each of the nanofibers was measured, and the number average value was taken as the average fiber diameter (D).

  The average fiber length (L) was determined as follows. The nanofiber dispersion to be evaluated was diluted with pure water to 100 ppm, and the nanofibers were uniformly dispersed using an ultrasonic cleaner. This nanofiber dispersion was cast onto a silicon wafer whose surface had been hydrophilized in advance using concentrated sulfuric acid, and dried at 110 ° C. for 1 hour to prepare a sample. Using an image of the obtained sample observed with a scanning electron microscope (SEM, JSM-7400F) (2,000 ×) manufactured by Nippon Denshi Co., Ltd., the number of samples: 150 to 250, one for each nanofiber. The fiber length of the book was measured, and the number average value was taken as the average fiber length (L).

In the nanofibers used in the present invention, when mixed with the liquid medium, the nanofibers are uniformly dispersed in the liquid while maintaining the primary fiber diameter, and cells and / or tissues are not substantially increased in the viscosity of the liquid. Substantially holds it and has the effect of preventing its settling. Not substantially increasing the viscosity of the liquid means that the viscosity of the liquid does not exceed 8 mPa · s. At this time, the viscosity of the liquid (that is, the viscosity of the medium composition produced by the production method of the present invention) is 8 mPa · s or less, preferably 4 mPa · s or less, more preferably 2 mPa · s or less. It is. Furthermore, when the nanofibers are dispersed in a liquid culture medium, they exhibit an effect of uniformly suspending (preferably suspending) cells and / or tissues without substantially increasing the viscosity of the liquid. For example, the chemical structure, molecular weight, physical properties and the like of the nanofibers are not limited at all.
The viscosity of the liquid containing nanofibers can be measured, for example, by the method described in the examples below. Specifically, it can be evaluated using tuning fork vibrational viscosity measurement (SV-1A, A & D Company Ltd.) under 25 ° C. conditions.

Examples of raw materials that constitute the nanofibers include, but are not limited to, low molecular weight compounds and high molecular weight compounds.
Preferred specific examples of the low molecular weight compounds used in the present invention include, but are not limited to, for example, L-isoleucine derivatives, L-valine derivatives, amino acid derivatives such as L-lysine derivatives, etc .; Cyclohexanediamine derivatives such as diaminocyclohexanediamide derivatives, 5-aminoisophthalic acid derivatives, R-12-hydroxystearic acid, 1,3,5-benzenetricarboxamide, cis-1,3,5-cyclohexanetricarboxamide, 2 Mention may be made of low molecular weight gelling agents such as 4-dibenzylidene-D-sorbitol, N-lauroyl-L-glutamic acid-α, γ-bis-n-butylamide, calcium dehydroabietic acid and the like.

  Preferred specific examples of the polymer compound used in the present invention include, but not particularly limited to, polysaccharides, polypeptides and the like.

  The polysaccharide means a glycopolymer in which 10 or more monosaccharides (eg, triose, tetrose, pentose, hexose, heptose, etc.) are polymerized. Polysaccharides include water insoluble polysaccharides and water soluble polysaccharides.

  Examples of non-water soluble polysaccharides include, but are not limited to, celluloses such as cellulose and hemicellulose; chitins such as chitin and chitosan; and the like.

The water-soluble polysaccharides include acidic polysaccharides having an anionic functional group. The acidic polysaccharide having an anionic functional group is not particularly limited, but, for example, a polysaccharide having a uronic acid (for example, glucuronic acid, iduronic acid, galacturonic acid, mannuronic acid) in the structure; Examples thereof include polysaccharides having phosphoric acid, and polysaccharides having a structure of both. More specifically, hyaluronic acid, gellan gum, deacylated gellan gum (DAG), rhamsan gum, diutatan gum, xanthan gum, carrageenan, xanthan gum, hexuronic acid, fucoidan, pectin, pectic acid, pectinic acid, heparan sulfate, heparin, heparitin What is comprised from 1 type, or 2 or more types from the group which consists of a sulfuric acid, kerato sulfate, chondroitin sulfate, dermatan sulfate, rhamnan sulfate, alginic acid, and those salts is illustrated.
As the salts mentioned herein, salts of alkali metals such as lithium, sodium and potassium; salts of alkaline earth metals such as calcium, barium and magnesium; salts of aluminum, zinc, copper, iron and the like; ammonium salts; tetraethylammonium and tetrabutyl Quaternary ammonium salts such as ammonium, methyltributylammonium, cetyltrimethylammonium, benzylmethylhexyldecylammonium and choline; with pyridine, triethylamine, diisopropylamine, ethanolamine, diolamine, tromethamine, meglumine, procaine, chlorprocaine etc. Salts and salts with amino acids such as glycine, alanine, valine and the like can be mentioned.

  The polypeptide includes polypeptides that constitute fibers in the living body. Specific examples include, but are not limited to, collagen, elastin, myosin, keratin, amyloid, fibroin, actin, tubulin and the like.

  As a raw material constituting the nanofibers used in the present invention, not only substances of natural origin but also substances produced by microorganisms, substances produced by genetic engineering, or artificially synthesized using enzymes or chemical reactions Substances are also included. The raw material constituting the nanofibers used in the present invention is preferably a substance of natural origin (ie, a substance extracted from nature) or a substance obtained by modifying it by a chemical reaction or an enzymatic reaction.

  In one aspect, the polysaccharide is a water insoluble polysaccharide. Preferred non-water soluble polysaccharides include cellulose; chitin such as chitin and chitosan. Cellulose and chitin are most preferred in view of the ability to lower the viscosity of the medium composition and the ease of recovery of cells or tissues.

  Cellulose is a natural polymer compound in which D-glucopyranose, which is a six-membered ring of glucose, is β-1,4 glucosidically linked. As raw materials, for example, wood, bamboo, hemp, jute, kenaf, cotton, cellulose derived from plants such as agricultural products and food residues, or bacterial cellulose, shiogusa (cladophora), gray plant (glaucochtis), baronia, hoyacellulose etc. Cellulose produced or produced animal can be used. In plant-derived cellulose, very thin fibers called microfibrils are further bundled to form a higher-order structure in stages with fibrils, lamellae and fiber cells. In bacterial cellulose, microfibrils of cellulose secreted from bacterial cells form a fine network structure with the same thickness.

  In the present invention, high purity cellulose raw materials such as cotton and bacterial cellulose can be used as raw materials, but it is preferable to use isolated and purified celluloses derived from plants other than this. Cellulose preferably used in the present invention is cotton cellulose, bacterial cellulose, kraft pulp cellulose, microcrystalline cellulose or the like. In particular, kraft pulp cellulose is preferably used because it has a high floating action.

  Chitin refers to one or more carbohydrates selected from the group consisting of chitin and chitosan. The main sugar units constituting chitin and chitosan are N-acetylglucosamine and glucosamine, respectively, and in general, chitin and glucosamine having a high content of N-acetylglucosamine and being poorly soluble in an acidic aqueous solution What has a high content and is soluble in an acidic aqueous solution is chitosan. In the present specification, for convenience, those in which the ratio of N-acetylglucosamine in constituent sugars is 50% or more are referred to as chitin, and those in which the ratio of less than 50% is referred to as chitosan. From the viewpoint of achieving a high floating action, the higher the ratio of N-acetylglucosamine to the sugar unit constituting chitin, the better. The ratio of N-acetylglucosamine to the sugar unit constituting chitin is preferably 80% or more, more preferably 90% or more, still more preferably 98% or more, and most preferably 100%.

  As a raw material of chitin, many biological resources can be used, such as shrimp, crab, insect, shellfish, and mushroom, for example. The chitin used in the present invention may be a chitin having an α-type crystal structure such as chitin derived from crab shells or shrimp shells, or a chitin having a β-type crystal structure such as chitin derived from squid shell It is also good. Crab and shrimp shells are often treated as industrial waste, and they are preferable as raw materials from the viewpoint of easy availability and effective use, but they are deproteinized and deashed to remove proteins and ash contained as impurities. A process is required. Therefore, in the present invention, it is preferable to use purified chitin which has been subjected to the dematrixing treatment. Purified chitin is commercially available.

  In one aspect, the polysaccharide is a water soluble polysaccharide. Preferred water-soluble polysaccharides include deacylated gellan gum, carrageenan and the like. Deacylated gellan gum is most preferred from the viewpoint of achieving high flotation.

  A deacylated gellan gum is a linear high chain structure composed of four sugars of 1-3-linked glucose, 1-4-linked glucuronic acid, 1-4-linked glucose and 1-4-linked rhamnose as structural units It is a molecular polysaccharide, and in general formula (I) below, R 1 and R 2 are both hydrogen atoms, and n is a polysaccharide represented by an integer of 2 or more. However, although R1 may contain a glyceryl group and R2 may contain an acetyl group, the content of acetyl group and glyceryl group is preferably 10% or less, more preferably 1% or less.

As a method for producing gellan gum, the produced microorganism may be cultured in a fermentation medium, mucosal substances produced outside the bacteria may be recovered by a conventional purification method, and after drying, crushing and the like, it may be powdered. In addition, deacylated gellan gum is used to culture a microorganism that produces gellan gum in a fermentation medium, and to recover mucous membrane products produced outside the fungus. At this time, the mucous membrane may be subjected to an alkali treatment to deacylate the glyceryl group and acetyl group bound to the 1-3-linked glucose residue and then recovered. Purification of the deacylated gellan gum from the collected mucous membrane material includes, for example, liquid-liquid extraction, fractional precipitation, crystallization, various ion exchange chromatography, gel filtration chromatography using Sephadex LH-20, etc., activated carbon Adsorption / desorption treatment of the active substance by adsorption chromatography with silica gel etc. or thin layer chromatography, or high performance liquid chromatography using a reverse phase column, etc. alone or in combination in any order and repeated use can do. Examples of gellan gum-producing microorganisms include, but are not limited to, Sphingomonas elodea and microorganisms in which the genes of the microorganisms are modified.
And, in the case of deacylated gellan gum, commercially available ones, for example, "KELCOGEL (registered trademark of CPI-Kelco" CG-LA "manufactured by Sanyo Co., Ltd .; -It is possible to use Kelco's registered trademark) and the like.

  The weight average molecular weight of the polymer compound used in the present invention is preferably 1,000 to 50,000,000, more preferably 10,000 to 20,000,000, and still more preferably 100,000 to 10,000. , 000. For example, the molecular weight can be estimated from polyethylene glycol by gel permeation chromatography (GPC), pullulan conversion, viscosity of an aqueous solution, and the like.

  In the present invention, the above-described polymer compounds can be used in combination of two or more (preferably two). The type of combination of the macromolecular compounds forms nanofibers in the medium composition or disperses as nanofibers to suspend cells and / or tissues without substantially increasing the viscosity of the liquid medium (preferably, The combination is not particularly limited as long as it can be suspended and allowed to stand, but preferably, the combination comprises at least cellulose, chitin, collagen, or deacylated gellan gum. That is, suitable combinations of polymer compounds include cellulose, chitin, collagen, or deacylated gellan gum; and other polymer compounds (eg, xanthan gum, alginic acid, carrageenan, diutan gum, methylcellulose, locust bean gum or Their salts) are included.

[Preparation of nanofibers]
The medium composition of the present invention comprises nanofibers prepared from the above-mentioned raw materials. The preparation method of nanofibers is the case where a water-insoluble polymer compound (eg, water-insoluble polysaccharide such as cellulose, chitin, etc.) is used as a raw material, and a water-soluble polymer compound (eg, deacylated gellan gum etc.) This is different from the case of using water-soluble polysaccharides of

  When the raw material of nanofibers is a non-water-soluble polymer compound (for example, non-water-soluble polysaccharides such as cellulose and chitin), nanofibers are usually obtained by grinding the raw materials. The grinding method is not limited, but in order to reduce the fiber diameter and length to be described later to meet the purpose of the present invention, a strong shear force such as a medium stirring mill such as a high pressure homogenizer, grinder (stone mill) or bead mill can be obtained. Methods are preferred.

  Among these, it is preferable to miniaturize using a high pressure homogenizer, and for example, it is desirable to miniaturize (pulverize) using a wet pulverizing method as disclosed in, for example, Japanese Patent Application Laid-Open No. 2005-270891 and Patent No. 5232976. . Specifically, the raw materials are crushed by injecting dispersions in which the raw materials are dispersed at a high pressure from a pair of nozzles and causing them to collide, and for example, a star burst system (manufactured by Sugino Machine Co., Ltd.) This can be carried out by using a high pressure crusher) or Nanoveita (a high pressure crusher of Yoshida Kikko Co., Ltd.).

  When refining (grinding) raw materials using the above-mentioned high-pressure homogenizer, the degree of refinement and homogenization is determined by the pressure for pumping to the ultra-high pressure chamber of the high-pressure homogenizer and the number of times of passing through the ultra-high pressure chamber (number of times of processing) And the concentration of the raw material in the aqueous dispersion. The pumping pressure (processing pressure) is usually 50 to 250 MPa, preferably 150 to 245 MPa. If the pumping pressure is less than 50 MPa, miniaturization of the nanofibers may be insufficient, and the expected effect may not be obtained by the miniaturization.

  Moreover, the density | concentration of the raw material in the water dispersion liquid at the time of refinement | miniaturization processing is 0.1 mass%-30 mass%, Preferably it is 1 mass%-10 mass%. If the concentration of the raw material in the aqueous dispersion is less than 0.1% by mass, the productivity is low, and if the concentration is higher than 30% by mass, the pulverization efficiency is low and desired nanofibers can not be obtained. The number of times of the treatment of pulverization (pulverization) is not particularly limited, and it depends on the concentration of the raw material in the aqueous dispersion, but the treatment number of times is 10 to 10 when the concentration of the raw material is 0.1 to 1% It can be sufficiently refined in about 100 times, but in 1 to 10% by mass, about 10 to 1000 times are required. In addition, when the concentration is higher than 30% by mass, it is unrealistic from an industrial point of view because several thousand or more processing times are required and the viscosity increases to such an extent that the handling is hindered. .

  When the raw material of nanofibers is a water-soluble polymer compound (for example, water-soluble polysaccharide such as deacylated gellan gum), when the substance is added to the medium, the substance is via the metal cation in the medium Assembling and forming nanofibers in the culture medium and constructing a three-dimensional network results in the formation of nanofibers in which cells or tissues can be suspended and cultured.

The concentration of nanofibers in the medium composition of the present invention should be set appropriately so that cells and / or tissues can be suspended (preferably suspended) without substantially increasing the viscosity of the medium. However, it is usually from 0.0001% to 1.0% (w / v), for example from 0.0005% to 1.0% (w / v), preferably from 0.001% to 0.5% (w / v). The weight / volume) is more preferably in the range of 0.005% to 0.1% (weight / volume), still more preferably 0.005% to 0.05% (weight / volume).
For example, in the case of cellulose nanofibers, usually 0.0001% to 1.0% (weight / volume), for example 0.0005% to 1.0% (weight / volume), preferably 0.001% to 0.5 % (W / v), more preferably 0.01% to 0.1% (w / v), more preferably 0.01% to 0.05% (w / v) in the medium .
In the case of pulp cellulose nanofibers among cellulose nanofibers, the lower limit value of the concentration in the culture medium is preferably 0.01% (weight / volume) from the viewpoint of floating action expression and from the viewpoint of enabling suspension stationary culture. Or higher), 0.015% (w / v), 0.02% (w / v), 0.025% (w / v) or more, or 0.03% (w / v) or more. . In the case of pulp cellulose nanofibers, the upper limit value of the concentration in the medium is preferably 0.1% (weight / volume) or less, or 0.04% (weight) from the viewpoint of not substantially increasing the viscosity of the medium. / Capacity) or less.
In the case of microcrystalline cellulose nanofibers, the lower limit value of the concentration in the culture medium is preferably 0.01% (weight / volume) or more, 0.03% (weight / volume) or more, from the viewpoint of floating action expression. .05% (weight / volume) or more. From the viewpoint of enabling suspension and stationary culture, the lower limit value of the concentration of microcrystalline cellulose nanofibers in the medium is preferably 0.03% (weight / volume) or more, or 0.05% (weight / volume) or more It is. In the case of microcrystalline cellulose nanofibers, the upper limit value of the concentration in the medium is preferably 0.1% (weight / volume) or less.
In the case of chitin nanofibers, generally 0.0001% to 1.0% (weight / volume), for example 0.0005% to 1.0% (weight / volume), preferably 0.001% to 0.5% (weight / volume). W / v), more preferably 0.01% to 0.1% (w / v), most preferably 0.03% to 0.07% (w / v) medium may be added. From the viewpoint of floating action expression, the lower limit value of chitin nanofiber concentration in the medium is preferably 0.0001% (weight / volume) or more, 0.0003% (weight / volume) or more, 0.0005% (weight / volume). Or higher, or 0.001% (w / v) or higher. From the viewpoint of enabling suspension and stationary culture, the lower limit value of chitin nanofibers in the medium is preferably 0.03% (weight / volume) or more. The upper limit of chitin nanofiber concentration in the medium is preferably 0.1% (weight / volume) or less.
In the case of water-insoluble nanofibers such as cellulose nanofibers and chitin nanofibers, the concentration of 0.1% (weight / volume) or less usually does not substantially increase the viscosity of the medium composition.
In the case of carrageenan, 0.0005% to 1.0% (weight / volume), preferably 0.001% to 0.5% (weight / volume), more preferably 0.01% to 0.1% (weight) / Volume), most preferably 0.02% to 0.1% (weight / volume) medium. The lower limit value of the carrageenan concentration in the medium is preferably 0.01% or more from the viewpoint of floating action expression and the viewpoint of enabling floating stationary culture. The upper limit of carrageenan concentration in the culture medium is preferably 0.1% (weight / volume) or less. From the viewpoint of not substantially increasing the viscosity of the medium, it is also preferable to set the upper limit of carrageenan to 0.04% (weight / volume) or less.
In the case of deacylated gellan gum, it is usually 0.001% to 1.0% (weight / volume), for example, 0.005% to 1.0% (weight / volume), preferably 0.003% to 0.5. % (W / v), more preferably 0.01% to 0.1% (w / v), more preferably 0.01 to 0.05% (w / v), most preferably 0.01% To 0.02% (weight / volume) medium may be added. From the viewpoint of expression of floating action, the lower limit value of the deacylated gellan gum concentration in the medium is preferably 0.005% (weight / volume) or more, or 0.01% or more. The lower limit value of the deacylated gellan gum concentration in the culture medium is preferably 0.01% (weight / volume) or more from the viewpoint of enabling suspension stationary culture. From the viewpoint of not substantially increasing the viscosity of the culture medium, the upper limit value of the deacylated gellan gum concentration in the culture medium is 0.05% (weight / volume) or less. From the viewpoint of not substantially increasing the viscosity of the culture medium, it is also preferable to set the upper limit value of the deacylated gellan gum to 0.04% (w / v) or less.

[Combined use of polysaccharides]
In addition to the above-mentioned nanofibers, multiple types (preferably two types) of polysaccharides can be used in combination. The concentration of the polysaccharide can be set as appropriate as long as cells and / or tissues can be uniformly suspended (preferably suspended and allowed to stand) without substantially increasing the viscosity of the liquid culture medium. For example, when using a combination of nanofibers and polysaccharides, the concentration of nanofibers is 0.005 to 0.1% (weight / volume), preferably 0.01 to 0.07% (weight / volume). The concentration of the polysaccharide is, for example, 0.005 to 0.4% (w / v), preferably 0.1 to 0.4% (w / v). Specific combinations of concentration ranges are exemplified below.
Cellulose or chitin nanofibers: 0.005 to 0.1% (preferably 0.01 to 0.07%) (weight / volume)
Polysaccharide xanthan gum: 0.1 to 0.4% (weight / volume)
Sodium alginate: 0.1 to 0.4% (weight / volume) (preferably 0.0001 to 0.4% (weight / volume))
Locust bean gum: 0.1 to 0.4% (weight / volume)
Methylcellulose: 0.1 to 0.4% (weight / volume) (preferably 0.2 to 0.4% (weight / volume))
Carrageenan: 0.05 to 0.1% (weight / volume)
Daiyutan gum: 0.05 to 0.1% (weight / volume)
Native gellan gum: 0.0001 to 0.4% (weight / volume)

The concentration can be calculated by the following equation.
Concentration (%) = weight of nanofibers (g) / volume of medium composition (ml) × 100

[Metal cation]
In one aspect, the medium composition of the present invention contains a metal cation, such as a divalent metal cation (such as calcium ion, magnesium ion, zinc ion, iron ion and copper ion), preferably calcium ion. In particular, when the nanofiber contained in the medium composition of the present invention is composed of a water-soluble polymer compound (for example, a water-soluble polysaccharide such as deacylated gellan gum), the medium composition of the present invention is It is preferred to contain a metal cation. By containing the metal cation, the water-soluble polymer compound (for example, water-soluble polysaccharide such as deacylated gellan gum) assembles via the metal cation to form nanofibers in the medium composition, Constructing a three-dimensional network results in the formation of nanofibers that can be suspended and cultured in cells or tissues.

[Culture medium]
Examples of the medium contained in the medium composition of the present invention include Dulbecco's Modified Eagles Medium (DMEM), Ham's F12 Medium (Ham's Nutrient Mixture F12), DMEM / F12 Medium, McCoy 5A medium (McCoy's 5A medium), Eagle MEM medium (Eagles's Minimum Essential Medium; EMEM), αMEM medium (alpha Modified Eagles's Minimum Essential Medium; αMEM), MEM medium (Minimum Essential Medium), RPMI 1640 medium Iscove's Modified Dulbecco '(Iscove's Modified Dulbecco'Medium; IMDM), MCDB 131 medium, William medium E, IPL 41 medium, Fischer's medium, StemPro 34 (manufactured by Invitrogen), X-VIVO 10 (manufactured by Kenbrex), X-VIVO 15 (manufactured by Kenbrex), HPGM (Kenbrecks), StemSpan H3000 (Stemcell Technology), StemSpanSFEM (Stemcell Technology), Stemline II (Sigma Aldrich), QBSF-60 (Quality Biological), StemProhESCSFM (Invitrogen), mTeSR1 or 2 medium (manufactured by Stem Cell Technology), Sf-900 II (manufactured by Invitrogen), Opti-Pro (manufactured by Invitrogen), Etc., and the like.

  When cells and / or tissues are of plant origin, Murashige-Skoog (MS) medium, Linsmeier-Skoog (LS) medium, white medium, Gambog B5 medium, Nitsche medium, Herler medium, Morel usually used for plant tissue culture Plant growth regulators such as auxins and cytokinins as necessary in a basic medium such as medium or a modified medium (for example, the ammonia nitrogen concentration is halved) in which these medium components are corrected to an optimum concentration A medium to which (plant hormone) is added at an appropriate concentration may be mentioned as the medium. These media can be further supplemented with caseinolytic enzymes, corn steep liquor, vitamins and the like, as necessary. Examples of auxins include 3-indoleacetic acid (IAA), 3-indolebutyric acid (IBA), 1-naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), etc. Not limited to them. Auxins, for example, can be added to the culture medium at a concentration of about 0.1 to about 10 ppm. Examples of cytokinins include, but are not limited to, kinetin, benzyl adenine (BA), zeatin and the like. Cytokinins can be added to the medium, for example, at a concentration of about 0.1 to about 10 ppm.

  Those skilled in the art may freely add sodium, potassium, calcium, magnesium, phosphorus, chlorine, various amino acids, various vitamins, antibiotics, serum, fatty acids, sugars and the like to the above-mentioned culture medium according to the purpose. In the case of animal-derived cell and / or tissue culture, one skilled in the art can add one or more other chemical components or biological components in combination depending on the purpose. Ingredients to be added to the medium of cells and / or tissues derived from animals include fetal bovine serum, human serum, horse serum, insulin, transferrin, lactoferrin, cholesterol, ethanolamine, sodium selenite, monothioglycerol, 2- Mercaptoethanol, bovine serum albumin, sodium pyruvate, polyethylene glycol, various vitamins, various amino acids, agar, agarose, collagen, methylcellulose, various cytokines, various hormones, various growth factors, various extracellular matrices, various cell adhesion molecules, etc. Be Examples of cytokines added to the medium include interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), Interleukin-5 (IL-5), Interleukin-6 (IL-6), Interleukin-7 (IL-7), Interleukin-8 (IL-8), Interleukin-9 (IL-9), Interleukin-10 (IL-10), Interleukin-11 (IL-11), Interleukin-12 (IL-12), Interleukin-13 (IL-13), Interleukin-14 (IL-14), Interleukin-15 (IL-15), Interleukin-18 (IL-18), Interleukin-21 (IL-21), Interferon-α (IFN-α), Interferon-β (IFN-β), Interferon-γ (IFN-γ), Granulocyte Colony Stimulating Factor (G-CSF), Monocyte Colony Stimulating Factor (M-CSF), Granules Sphere-macrophage colony stimulating factor (GM-CSF), stem cell factor (SCF), flk2 / flt3 ligand (FL), leukemia cell inhibitory factor (LIF), oncostatin M (OM), erythropoietin (EPO), thrombopoietin (TPO) And the like, but not limited thereto.

  Hormones added to the medium include melatonin, serotonin, thyroxine, triiodothyronine, epinephrine, norepinephrine, dopamine, anti-Mueller's tube hormone, adiponectin, adrenocorticotropic hormone, angiotensinogen and angiotensin, antidiuretic hormone, atria Natriuretic peptide, calcitonin, cholecystokinin, corticotropin releasing hormone, erythropoietin, follicle stimulating hormone, gastrin, ghrelin, glucagon, gonadotropin releasing hormone, growth hormone releasing hormone, human chorionic gonadotropin, human placental lactogen, growth hormone , Inhibin, insulin, insulin-like growth factor, leptin, luteinizing hormone, melanocyte stimulating hormone, oxytocin, parathyroid hormone Prolactin, secretin, somatostatin, thrombopoietin, thyroid stimulating hormone, thyrotropin releasing hormone, cortisol, aldosterone, testosterone, dehydroepiandrosterone, androstenedione, dihydrotestosterone, estradiol, estrone, estriol, progesterone, calcitriol, calcidiol , Prostaglandin, leukotriene, prostacyclin, thromboxane, prolactin releasing hormone, lipotropin, brain natriuretic peptide, neuropeptide Y, histamine, endothelin, pancreatic polypeptide, renin, and enkephalin, but is not limited thereto is not.

As growth factors to be added to the medium, transforming growth factor-α (TGF-α), transforming growth factor-β (TGF-β), macrophage inflammation protein-1α (MIP-1α), epidermal growth factor ( EGF), fibroblast growth factor-1, 2, 3, 4, 5, 6, 7, 8 or 9 (FGF-1, 2, 3, 4, 5, 6, 7, 8, 9), nerve Cell growth factor (NGF) hepatocyte growth factor (HGF), leukemia inhibitory factor (LIF), protease nexin I, protease nexin II, platelet derived growth factor (PDGF), cholinergic differentiation factor (CDF), chemokine, Notch ligand (such as Delta1), Wnt protein, angiopoietin-like protein 2, 3, 5 or 7 (Angpt 2, 3, 5, 7), insulin-like growth factor (IGF) , Insulin-like growth factor binding protein (IGFBP), but such pleiotrophin (Pleiotrophin), and the like, but is not limited to these.
Also, artificially modified amino acid sequences of these cytokines and growth factors can be added by genetic recombination techniques. Examples thereof include IL-6 / soluble IL-6 receptor complex or Hyper IL-6 (a fusion protein of IL-6 and soluble IL-6 receptor).

  Examples of various extracellular matrices and various cell adhesion molecules include collagen I to XIX, fibronectin, vitronectin, laminin-1 to 12, nitrogen, tenascin, thrombospondin, von Willebrand factor, osteopontin, fibrinogen, Various elastins, various proteoglycans, various cadherins, desmocollins, desmogleins, various integrins, E-selectin, P-selectin, L-selectin, immunoglobulin superfamily, matrigel, poly-D-lysine, poly-L-lysine, chitin, Chitosan, sepharose, hyaluronic acid, alginic acid gel, various hydrogels, and fragments thereof are mentioned.

  Examples of antibiotics to be added to the medium include sulfa preparations, penicillin, pheneticillin, methicillin, oxacillin, oxacillin, cloxacillin, fulcloxacillin, flucloxacillin, nafcillin, ampicillin, penicillin, amoxicillin, cyclacillin, ticarcillin, piperacillin, azurocilin, and the like. Mexulacilin, mesilinam, andinocillin, cephalosporin and derivatives thereof, oxophosphoric acid, amifloxacin, temafloxacin, nalidixic acid, nalidixic acid, piromidic acid, ciprofloxane, cinoxacin, norfloxacin, perfloxacin, rosoxacin, ofloxacin, enoxacin, pipemidic acid, sulbactam, clavulacitric acid , Β-bromopenicillanic acid, β-chloropenicillanic acid, 6-acetylmethylene-penicillanic acid, Formaldehyde food ester of foxazole, sultanpicillin, adinocillin and sulbactam, tazobactam, aztreonam, sulfazetin, isosulfasetin, norcadicin, m-carboxyphenyl, methyl phenylacetamidophosphonate, chlortetracycline, oxytetracycline, tetracycline, dememe It includes crocycline, doxycycline, metacycline, as well as minocycline.

[Method of producing medium composition]
The cells and / or tissues are cultured to a concentration that allows the cells and / or tissues to be uniformly suspended (preferably suspended) without substantially increasing the viscosity of the liquid medium. The culture medium composition of the present invention can be produced by mixing with the culture medium used for the preparation. The present invention also provides a method of producing such a medium composition of the present invention.

  The shape of the nanofibers may be in the form of powders, tablets, pills, capsules, formulated solids such as granules, liquids such as dispersions in suitable physiological aqueous solvents, or substrates or substrates It may be in a state of being Additives at the time of formulation include preservatives such as p-hydroxybenzoic acid esters; excipients such as lactose, glucose, sucrose and mannitol; lubricants such as magnesium stearate and talc; polyvinyl Binders such as alcohol, hydroxypropyl cellulose and gelatin; surfactants such as fatty acid esters; and plasticizers such as glycerin. These additives are not limited to those described above, and can be freely selected by those skilled in the art if available. The sterilization method is not particularly limited, and examples thereof include radiation sterilization, ethylene oxide gas sterilization, autoclave sterilization, filter sterilization and the like.

  In a preferred embodiment, the medium composition of the present invention is prepared by mixing a dispersion of the nanofibers in a physiological aqueous solvent with a liquid medium. The dispersion may be sterilized (autoclave, gamma sterilization, etc.). Alternatively, the dispersion and a liquid medium (aqueous solution of medium) prepared by dissolving powdered medium in water may be mixed and then sterilized. Sterilization of the dispersion and liquid medium may be performed separately prior to mixing. Examples of aqueous solvents include, but are not limited to, water, dimethylsulfoxide (DMSO), and the like. Water is preferred as the aqueous solvent. The aqueous solvent may contain suitable buffers and salts. The dispersion of nanofibers is useful as a culture medium additive for preparing the culture medium composition of the present invention. The present invention also provides such media additives.

  The mixing ratio of the dispersion of nanofibers: liquid medium (aqueous solution of medium) is usually 1:99 to 99: 1, preferably 10:90 to 90:10, more preferably 20:80 to 80:20. is there.

When the nanofibers are composed of a water-soluble polymer compound (for example, a water-soluble polysaccharide such as deacylated gellan gum), the water-soluble compound is used instead of mixing the nanofibers with the medium. The medium composition of the present invention may be produced by mixing the medium with a high molecular weight compound (for example, a water-soluble polysaccharide such as deacylated gellan gum) and the medium to form nanofibers in the medium. . The form of the polymer compound may be a powder, a tablet, a pill, a capsule, a formulated solid such as granules, a liquid such as a solution or suspension dissolved with a suitable solvent and a solubilizer, or Or bound to a carrier. Additives at the time of formulation include preservatives such as p-hydroxybenzoic acid esters; excipients such as lactose, glucose, sucrose and mannitol; lubricants such as magnesium stearate and talc; polyvinyl Binders such as alcohol, hydroxypropyl cellulose and gelatin; surfactants such as fatty acid esters; and plasticizers such as glycerin. These additives are not limited to those described above, and can be freely selected by those skilled in the art if available.
In addition, the above-mentioned polymer compound may be subjected to a sterilization treatment, if necessary. The sterilization method is not particularly limited, and examples thereof include radiation sterilization, ethylene oxide gas sterilization, autoclave sterilization, filter sterilization and the like.

  In a preferred embodiment, an aqueous solution of a water-soluble polymer compound (for example, a water-soluble polysaccharide such as deacylated gellan gum) (which is referred to as culture medium additive 2) is used in the production method of the present invention. The aqueous solution can be obtained by dissolving a solid (eg, powder) of a water-soluble polymer compound in a physiological aqueous solvent. Examples of aqueous solvents include, but are not limited to, water, dimethylsulfoxide (DMSO), and the like. Water is preferred as the aqueous solvent.

  The aqueous solvent may contain suitable buffers and salts. The aqueous solvent may or may not contain a divalent metal cation, but in a preferred embodiment, does not contain a divalent metal cation. When the aqueous solvent does not contain a divalent metal cation, a water-soluble polymer compound (for example, a water-soluble polysaccharide such as deacylated gellan gum) in the aqueous solution can be a nanofiber capable of suspending and culturing cells or tissues. This is because it is difficult to form and stable storage in a state of being dissolved in water.

  The medium additive may further contain an additive that enhances the effect of nanofibers or lowers the concentration at the time of use. As examples of such additives, one or more kinds of polysaccharides such as guar gum, tamarind gum, propylene glycol alginate, locust bean gum, gum arabic, tara gum, tamarind gum, methylcellulose and the like can be mixed.

  Although the method for producing the medium composition of the present invention is exemplified, the present invention is not limited thereby. The nanofibers are added to ion exchanged water or ultrapure water. After stirring at room temperature until the whole is dispersed uniformly, sterilization (for example, autoclave sterilization at 121 ° C. for 20 minutes) is performed. While stirring (for example, a homomixer etc.) any medium to be used for stationary culture, the sterilized nanofiber dispersion is added to the medium and mixed with the medium to be uniform. The mixing method of the aqueous solution and the culture medium is not particularly limited, and examples thereof include manual mixing such as pipetting, and mixing using an apparatus such as a magnetic stirrer, a mechanical stirrer, a homomixer, and a homogenizer.

  For example, when preparing a medium composition using cellulose nanofibers, 0.0001% to 5.0% (weight / volume), preferably 0.001% to 1.0% (weight / volume), more preferably The cellulose nanofibers are added to ion-exchanged water or ultrapure water so as to be 0.01% to 0.6% (weight / volume). Then, after stirring at room temperature until the whole becomes uniform, sterilization (for example, autoclave sterilization at 121 ° C. for 20 minutes) is performed. For example, while stirring a liquid culture medium such as DMEM culture medium with a homomixer etc., the aqueous solution is added to the culture medium to a desired final concentration (for example, when the final concentration is 0.03%, 0.6% aqueous solution: The medium ratio is 1:20) and mixed uniformly. Alternatively, a liquid medium such as DMEM medium is pipetted into the aqueous solution so as to obtain a desired final concentration (for example, when the final concentration is 0.03%, the ratio of 0.6% aqueous solution: medium is 1:20) Mix evenly by pipetting. The method of mixing the aqueous dispersion and the culture medium is not particularly limited, and examples thereof include manual mixing such as pipetting, and mixing using an apparatus such as a magnetic stirrer, a mechanical stirrer, a homomixer, and a homogenizer.

[Culture method]
The present invention relates to a culture method for growing cells or tissues using the above-mentioned medium composition of the present invention; a method for recovering the obtained cells or tissues by, for example, filtration, centrifugation or magnetic separation; The present invention also provides a method of producing a sphere using

  The nanofibers used in the present invention have an effect of suspending the cells and / or tissues in a liquid containing the nanofibers (preferably, by suspending the cells and / or tissues when the cells and / or tissues are cultured in vitro) Is an indicator of Due to the floating effect, cells and / or tissues per fixed volume can be expanded and cultured as compared to monolayer culture. In addition, when rotation and shaking are involved in the conventional suspension culture method, shear force is exerted on cells and / or tissues, resulting in low proliferation and recovery rates of cells and / or tissues, or impaired cell function. However, by using the medium composition containing the nanofibers of the present invention, it is possible to uniformly disperse cells and / or tissues without performing operations such as shaking, etc. Tissues can be obtained easily and in large quantities without loss of cellular function. In addition, when cells and / or tissues are suspended and cultured in a medium containing a conventional gel base, it may be difficult to observe and recover the cells and / or tissues, or the function may be impaired at the time of recovery. However, by using the medium composition containing the nanofiber of the present invention, cells and / or tissues can be suspended and cultured, and observed and recovered without impairing their functions. Moreover, although the medium containing the conventional gel base material may have high viscosity and it may be difficult to replace the medium, the medium composition containing the nanofibers of the present invention has a low viscosity, so it has a low viscosity. The medium can be easily replaced by using.

  The cells and / or tissues of human origin cultured by the method of the present invention can be transplanted for therapeutic purposes to patients with diseases or disorders. At this time, the type of disease or disorder to be treated, the pretreatment method and the cell transplantation method are appropriately selected by the party. Engraftment of the transplanted cells into the recipient and recovery from the disease or disorder, presence or absence of side effects associated with transplantation, and the effect of the treatment can be appropriately examined and judged by a general method in transplantation treatment.

  Furthermore, since the cells and / or tissues are efficiently grown, the medium composition of the present invention can be used as a reagent for cell research. For example, when elucidating factors that regulate differentiation and proliferation of cells and tissues, analyze changes in the number and type of cells, cell surface differentiation markers, and expressed genes when cocultured with cells and a desired factor, At this time, by using the medium composition of the present invention, the number of cells to be analyzed can not only be efficiently amplified, but also efficiently recovered. The culture conditions, culture apparatus, type of medium, type of nanofibers of the present invention, type of nanofibers, type of additives, type of additives, culture period, culture temperature etc. It is appropriately selected by the party from the range described in the specification. Cells grown or emerged by culture can be observed using a standard microscope in the art. At this time, the cultured cells may be stained using a specific antibody. The expressed gene altered by the factor of interest can be detected by extracting RNA (ribonucleic acid) from the cultured cells by Northern blotting, RT-PCR or the like. Moreover, a cell surface differentiation marker can be detected by ELISA or flow cytometry using a specific antibody, and the effect on differentiation or proliferation by a target factor can be observed.

  In addition, since cells and / or tissues are efficiently grown using the medium composition of the present invention, the culture method of the present invention is a method of growing cells and / or tissues or a method of promoting cell and / or tissue growth. As excellent. When cells and / or tissues are cultured using the medium composition of the present invention, the cells and / or tissues do not adhere to the culture vessel, and are not distributed unevenly on only the bottom of the culture vessel, and spread three-dimensionally. Disperse and promote proliferation. In particular, when chitin nanofibers are used as nanofibers, cells adhere to chitin nanofibers and grow strongly as a scaffold, and as a result, proliferated cells, cell clusters (spheres etc.) and / or tissues It becomes a state of being connected on the nanofiber in a tufted shape. This growth promoting effect includes the concentration of nanofibers in the medium composition that is sufficient for suspending cells and / or tissues (ie, avoiding adhesion of cells and tissues to the culture vessel). Well, suspension (that is, cells and / or tissues uniformly dispersed in the liquid medium composition and in a suspended state without external pressure, vibration, shaking, rotation, etc.) It is not essential that it is possible. For example, in the case of chitin nanofibers, a concentration of at least 0.0001% (weight / volume) sufficient for the expression of buoyancy causes 0.03% (weight / volume) to enable stable suspension and stationary culture. Even if the concentration is lower (eg, 0.025% (weight / volume) or less, 0.02% (weight / volume) or less), the growth promoting effect can be achieved. In the case of microcrystalline cellulose nanofibers, the concentration is less than 0.03% (weight / volume), which enables stable suspension / stationary culture if the content is 0.01% (weight / volume) or more sufficient for suspension expression Even in the case of (eg, 0.025% (weight / volume) or less, 0.02% (weight / volume) or less), the growth promoting effect is exhibited. In the case of deacylated gellan gum, the concentration is less than 0.01% (w / v) which enables stable suspension / stationary culture if it is 0.005% (w / v) or more sufficient for suspension expression For example, even if it is 0.009% (weight / volume) or less and 0.008% (weight / volume) or less), the growth promoting effect is exhibited.

  Among nanofibers, chitin nanofibers are particularly excellent in cell growth promoting effect.

  In the culture method of the present invention, any cells of floating cells and adherent cells can be used. Adherent cells are cells that require a scaffold for growth and proliferation. Floating cells are cells that do not require a scaffold for growth and proliferation. In the culture method of the present invention, preferably, adherent cells are used. In the method of the present invention, when adherent cells are used, adherent cells do not adhere to the bottom surface of the culture vessel, but are not dispersed only on the bottom surface of the culture vessel, dispersed with a three-dimensional spread and adhere to nanofibers Grow in state or sphere state. In particular, when chitin nanofibers are used as nanofibers, cells adhere to chitin nanofibers and grow strongly as a scaffold there, and as a result, proliferated cells and cell clusters (spheres etc.) become like grape bunches. It will be in a state of being connected on the nanofibers. Therefore, suspension culture of adherent cells becomes possible. In addition, as a result, the proliferation of adherent cells is promoted more than when cultured in a state of being adhered to the bottom of the culture vessel. In addition, adherent cells can be cultured at a higher density than in the case of culturing in a state of being adhered to the bottom of the culture vessel.

  In the culture method of the present invention, since the suspension culture of adherent cells is possible, after the adherent cells are suspended and cultured by the culture method of the present invention, the culture medium of the present invention is fresh without requiring removal of cells from the culture vessel. Adherent cells can be passaged simply by adding the composition to the culture after culture or by adding all or a part of the culture after culture to a fresh medium composition of the present invention. is there. The present invention also provides a method of subculturing such adherent cells. Therefore, by using the subculture method of the present invention, adherent cells can be subcultured without carrying out the detachment operation of the cells from the culture vessel. In addition, by using the subculture method of the present invention, the culture scale of adherent cells can be expanded without carrying out the detachment operation of the cells from the culture vessel. The detachment operation of cells from the culture vessel includes treatment with a chelating agent (eg, EDTA) and / or a proteolytic enzyme (eg, trypsin, collagenase). The passaging method of the present invention is a passaging of adherent cells (eg, adherent cells whose viability is reduced by the detaching operation, adherent cells whose trait is easily changed by the detaching operation) that are highly sensitive to the detaching operation It is advantageous to culture. Adherent cells sensitive to the detachment operation of cells from the culture vessel include human pluripotent stem cells; human precursor cells; primary cells prepared from tissues such as hepatocytes, kidney cells, chondrocytes, vascular cells and adipocytes; It includes, but is not limited to, cells that produce biopharmaceuticals (proteins for pharmaceuticals) such as MDCK cells, HEK 293 cells and CHO cells.

  The culture method of the present invention is useful for in vitro cell culture, because adherent cells can be cultured at high density and cells and / or tissues can be efficiently propagated using the medium composition of the present invention. Useful for the production of substances. The useful substance can be obtained by subjecting cells producing the useful substance to suspension culture in the medium composition of the present invention and isolating the useful substance from the culture. Useful substances include antibodies, enzymes (urokinase etc.), hormones (insulin etc.), cytokines (interferon, interleukin, tumor necrosis factor, colony stimulating factor, growth factor etc.), vaccine antigens, other physiologically active substances (proteins) , Peptides, etc.), but is not limited thereto. For cells that produce useful substances, non-transformed cells such as skin cells, chondrocytes, hepatocytes, pancreatic cells, and kidney cells, and genes encoding useful substances and genes involved in biosynthesis of useful substances have been introduced. Included are transformed cells. Cells producing useful substances may be adherent cells or floating cells, but are preferably adherent cells. The cells producing the useful substance are preferably cells that secrete the useful substance extracellularly. Specifically as cells producing useful substances, HEK293, CHO-K1, BHK-21, MDCK, Vero, HepG2, to which a gene encoding a useful substance or a gene involved in biosynthesis of a useful substance has been introduced. Although MCF-7 etc. can be mentioned, it is not limited to these. Cells used for the production of valuable substances such as recombinant proteins are well known to the person skilled in the art and these cells can be used in the method of the invention. When expanding the culture scale, is it possible to add a fresh medium composition of the present invention to the culture after culture without performing the detachment operation of cells from the culture vessel using the above-mentioned subculture method of the present invention? The whole or a part of the culture after culture may be added to a fresh medium composition of the present invention. Although it is necessary to remove the cells from the culture in order to isolate the useful substance from the culture, the medium composition of the present invention is not substantially enhanced in viscosity by the addition of nanofibers, and the cells Because they are suspended in the composition, cells can be removed by a simple method such as centrifugation or filtration. In addition, nanofibers in the medium composition can also be removed by a simple method such as centrifugation or filtration. Methods for isolating useful substances from culture are well known to those skilled in the art and include, for example, chromatography (eg, chromatography such as ion exchange chromatography, hydrophobic chromatography, affinity chromatography, reverse phase chromatography, etc.) Biochemical separation and purification methods of physiologically active substances are applicable.

  When culturing cells and / or tissues using the culture method of the present invention, petri dishes, flasks, plastic bags, Teflon bags, dishes, petri dishes, tissue cultures generally used for cell culture It is possible to culture using culture equipment such as dishes, multidishes, microplates, microwell plates, multiplates, multiwell plates, chamber slides, tubes, trays, culture bags, roller bottles and the like. The material of the culture equipment is not particularly limited, and examples thereof include glass, polyvinyl chloride, cellulose polymers, polystyrene, polymethyl methacrylate, polycarbonate, polysulfone, polyurethane, polyester, polyamide, polystyrene, polypropylene, and other plastics. . In addition, various surface treatments (for example, plasma treatment, corona treatment, etc.) may be applied to these plastics. Furthermore, these culture devices may be coated beforehand with extracellular matrix, cell adhesion molecules and the like. Such coating materials include collagen I to XIX, fibronectin, vitronectin, laminin-1 to 12, nitrogen, tenascin, thrombospondin, von Willebrand factor, osteopontin, fibrinogen, various elastins, various proteoglycans, Various cadherins, desmocollins, desmogleins, various integrins, E-selectin, P-selectin, L-selectin, immunoglobulins, hyaluronic acid, superfamily, matrigel, poly-D-lysine, poly-L-lysine, chitin, chitosan, These include sepharose, alginic acid gel, hydrogel, and fragments thereof. As these coating materials, those artificially modified in amino acid sequence by genetic recombination technology can also be used. Also, a coating material can be used to inhibit adhesion of cells and / or tissues to culture equipment. Such coating materials include, but are not limited to, silicon, poly (2-hydroxymethyl methacrylate), poly (2-methacryloyloxyethyl phosphoryl choline), and the like.

  For cell and / or tissue culture, cell seeding, medium exchange, cell image acquisition, culture cell recovery are automatically performed under mechanical control and in a closed environment to control pH, temperature, oxygen concentration, etc. However, it can also be performed by a bioreactor or an automatic culture device capable of culturing at high density. As a method of supplying new culture medium in the middle of culture using these devices and supplying required substances to cells and / or tissues without excess or deficiency, there are fed-batch culture, continuous culture and perfusion culture, whichever method Can also be used in the culture method of the present invention.

  The form and condition of cells and / or tissues cultured by the method of the present invention can be optionally selected by those skilled in the art. Preferred examples thereof include, but are not limited to, a state in which cells and / or tissues are dispersed alone in a medium composition, a state in which cells and / or tissues are adhered on a carrier surface, cells and / or Or a state in which the tissue is embedded in the carrier, a state in which a plurality of cells are aggregated to form a cell mass (sphere), or a state in which two or more types of cells are aggregated to form a cell mass (sphere), More preferably, cells and / or tissues adhere to the surface of the carrier, cells and / or tissues are embedded inside the carrier, a plurality of cells are aggregated to form a cell mass (sphere), or 2 More preferably, cells and / or tissues are adhered to the surface of the carrier, and a plurality of cells are aggregated to form a cell mass (sphere). Condition, or It can be mentioned state two or more of the cells to form a cell mass (spheres) collectively. Among these states, the state in which a cell mass (sphere) is formed is that cell-cell interactions and cell structures close to the in vivo environment are reconstructed, and culture can be performed with long-term maintenance of cell function. In addition, since it is relatively easy to recover cells, it can be mentioned as the most preferable state to be cultured by the method of the present invention.

  Carriers for supporting cells and / or tissues on the surface include microcarriers, glass beads, ceramic beads and the like composed of various polymers. Examples of the polymer include vinyl resin, urethane resin, epoxy resin, polystyrene, polymethyl methacrylate polyester, polyamide, polyimide, silicone resin, phenol resin, melamine resin, urea resin, aniline resin, ionomer resin, ionomer resin, polycarbonate, collagen, Dextran, gelatin, cellulose, alginate and mixtures thereof can be used. The carrier may be coated with a compound that enhances cell adhesion or enhances the release of substances from cells. Examples of such coating materials include poly (monostearoyl glyceride succinic acid), poly-D, L-lactide-co-glycolide, sodium hyaluronate, n-isopropylacrylamide, collagen I to XIX, fibronectin, vitronectin, laminin -1 to 12, nitrogen, tenascin, thrombospondin, von Willebrand factor, osteopontin, fibrinogen, various elastins, various proteoglycans, various cadherins, desmocholine, desmogleins, various integrins, E-selectin, P-selectin L-selectin, immunoglobulin superfamily, matrigel, poly-D-lysine, poly-L-lysine, chitin, chitosan, sepharose, algi Acid gel, various hydrogels, and the like In addition, these cleavage fragments. At this time, two or more kinds of coating materials may be combined. Furthermore, one kind of polysaccharide such as guar gum, tamarind gum, locust bean gum, gum arabic, tara gum, tamarind gum, methylcellulose and the like is used for the culture medium used for culturing the carrier having cells and / or tissues supported on the surface. The above can be mixed. The carrier may also contain a magnetic material, such as ferrite. The diameter of the carrier is several tens to several hundreds of μm, more preferably 100 to 200 μm, and the specific gravity thereof is preferably close to 1, more preferably 0.9 to 1.2, and particularly preferably about 1.0. is there. Examples of the carrier include, but are not limited to, Cytodex 1 (registered trademark), Cytodex 3 (registered trademark), Cytoline 1 (registered trademark), Cytoline 2 (registered trademark), Cytopore 1 (registered trademark), Cytopore 2 (registered trademark) Trademarks (above, GE Healthcare Life Sciences), Biosilon (registered trademark) (NUNC), Cultispher-G (registered trademark), Cultispher-S (registered trademark) (above, Thermo SCIENTIFIC), HILLEXCT (registered trademark), ProNectinF -COATED (registered trademark), and HILLEX II (registered trademark) (Solo Hill Engineering), and the like. The carrier may be sterilized if necessary. The sterilization method is not particularly limited, and examples thereof include radiation sterilization, ethylene oxide gas sterilization, autoclave sterilization and dry heat sterilization. There is no restriction | limiting in particular as a method to culture | cultivate an animal cell using the said support | carrier, The culture | cultivation method using a conventional fluidized bed type culture tank or a packed bed type culture tank can be used. At this time, the carrier having the cells and / or tissues supported on the surface can be uniformly dispersed without performing operations such as shaking by using the medium composition containing the nanofiber of the present invention. The cells and / or tissues of interest can be cultured without loss of cell function. The cells and / or tissues cultured by this method can be recovered by centrifugation or filtration while being supported by the carrier after the culture. At this time, after adding the liquid medium used, centrifugation or filtration may be performed. For example, the gravitational acceleration (G) at the time of centrifugation is 100 to 400 G, and the size of the pore of the filter used at the time of filtration is 10 μm to 100 μm, but is not limited thereto. In addition, if a magnetic material such as ferrite is included in the carrier, the cultured carrier can be recovered by magnetic force. The cells and / or tissues cultured by this method can be recovered by peeling them from the carrier using various chelating agents, heat treatments and enzymes.

  When cells and / or tissues are embedded in a carrier, materials composed of various polymers can be selected as the carrier. Examples of such polymers include collagen, gelatin, alginate, chitosan, agarose, polyglycolic acid, polylactic acid, fibrin adhesive, polylactic acid / polyglycolic acid copolymer, proteoglycan, glucosaminoglycan, polyurethane Sponges such as foam, DseA-3D (registered trademark), poly N-substituted acrylamide derivatives, poly N-substituted methacrylamide derivatives and copolymers thereof, polyvinyl methyl ether, polypropylene oxide, polyethylene oxide, polyvinyl alcohol partial acetate Temperature sensitive polymers, polyacrylamide, polyvinyl alcohol, methyl cellulose, nitrocellulose, cellulose butyrate, polyethylene oxide, poly (2-hydroxyethylmethacr . late) / polycaprolactone etc. hydrogel and the like. It is also possible to produce a carrier for embedding cells using two or more of these polymers. Furthermore, the carrier may have a physiologically active substance in addition to these polymers. Examples of the physiologically active substance include cell growth factor, differentiation inducing factor, cell adhesion factor, antibody, enzyme, cytokine, hormone, lectin, extracellular matrix and the like, and it is possible to contain a plurality of these. . Furthermore, it is possible to use a thickening medium such as guar gum, tamarind gum, propylene glycol alginate, locust bean gum, gum arabic, tara gum, methyl cellulose and the like for the medium used for culturing the carrier in which the cells and / or tissues are embedded. More than one species can be mixed.

  The method for embedding cells and / or tissues in these carriers is not particularly limited, but, for example, a mixture of cells and the above-mentioned polymer is drawn into a syringe and dropped into a medium via an injection needle of about 25G to 19G. Alternatively, a method such as dropping in a medium using a micropipette may be used. The size of the bead-like carrier formed here is determined by the shape of the device and the tip of the device used when dropping the cells and the polymer mixture, and is preferably several tens to several thousand μm, more preferably 100 to 2000 μm. The number of cells that can be cultured on a bead-like carrier is not particularly limited, but may be freely selected according to the bead size. For example, in the case of a bead-like carrier with a diameter of about 2000 μm, up to 5 million cells can be embedded in a bead-like carrier of this size. The cells may be dispersed one by one in the carrier or may form a cell mass in which a plurality of cells are aggregated. At this time, the carrier in which the cells and / or tissues are embedded can be uniformly dispersed without performing operations such as stirring by using the medium composition containing the nanofibers of the present invention. Cells and / or tissues can be cultured without loss of cell function. The cells and / or tissues cultured by this method can be recovered by centrifugation or filtration in a state embedded in a carrier after culture. At this time, after adding the liquid medium used, centrifugation or filtration may be performed. For example, the gravitational acceleration (G) at the time of centrifugation is 100 to 400 G, and the size of the pore of the filter used at the time of filtration is 10 μm to 100 μm, but is not limited thereto. Cells and / or tissues cultured by this method can be dispersed and recovered by decomposing the carrier using treatments such as various chelating agents, heat and enzymes.

  The method for forming cell aggregates (spheres) is not particularly limited and can be appropriately selected by those skilled in the art. Examples thereof include a method using a container having a cell non-adherent surface, a hanging drop method, a swirl culture method, a three-dimensional scaffold method, centrifugation, a method using aggregation by an electric field or a magnetic field, and the like. For example, for a method using a container having a cell non-adherent surface, cells of interest can be cultured in a surface-treated culture container that inhibits cell adhesion to form spheres. When this cell non-adherent culture vessel is used, first, the target cell is collected, and then the cell suspension is prepared, and the cell suspension is seeded and cultured. When culture is continued for about a week, cells spontaneously form spheres. As the non-cell-adhesive surface used at this time, a surface of a culture vessel such as a petri dish generally used may be coated with a substance that inhibits cell adhesion. Such substances include agarose, agar, copolymers of poly-HEMA (poly- (2-hydroxy-ethyl methacrylate)) 2-methacryloyloxyethyl phosphoryl choline and other monomers (such as butyl methacrylate), etc. However, without cytotoxicity, it is not limited thereto.

In addition, as a method of forming cell clumps (spheres), NATURE BIOTECHNOLOGY, VOL. 28, NO. 4, APRIL 2010, 361-366, NATURE PROTOCOLS, VOL. 6, NO. 5, 2011, 689-700, NATURE PROTOCOLS, VOL. 6, NO. 5, 2011, 572-579, Stem Cell Research, 7, 2011, 97-111, Stem Cell Rev and Rep, 6, 2010, 248-259 etc. can also be used.
In addition, the medium used in the culture to form the spheres can also contain components that accelerate the formation of the spheres or promote their maintenance. Examples of components having such an effect include dimethyl sulfoxide, superoxide dismutase, ceruloplasmin, catalase, peroxidase, L-ascorbic acid, L-ascorbic acid phosphate, tocopherol, flavonoid, uric acid, bilirubin, selenium-containing Examples thereof include ROCK inhibitors such as compounds, transferrin, unsaturated fatty acids, albumin, theophylline, forskolin, glucagon, dibutylyl cAMP, Y27632, Fasudil (HA1077), H-1152, Wf-536 and the like. As a selenium containing compound, sodium selenite, sodium selenate, dimethyl selenide, hydrogen selenide, selenomethionine, Se-methyl selenocysteine, selenocystathionine, selenocysteine, selenohomocysteine, adenosine-5'-phosphoselenic acid, Se-adenosylselenomethionine is mentioned. Moreover, in order to obtain a uniform cell aggregate of a target size, it is also possible to introduce a plurality of recesses having the same diameter as the target cell aggregate on the cell non-adhesive culture vessel to be used. When these recesses are in contact with each other or within the diameter of the target cell aggregate, the seeded cells do not form cell aggregates between the recess and the recess when the cells are seeded. It is possible to reliably form cell aggregates of a size corresponding to the volume in the recess and obtain a cell aggregate population of uniform size. The shape of the recess in this case is preferably a hemisphere or a cone.

Alternatively, spheres can be formed on a support having cell adhesion. Examples of such a support include collagen, polyrotaxane, polylactic acid (PLA), polylactic acid glycolic acid copolymer (PLGA), hydrogel and the like.
Spheres can also be formed by co-culture with feeder cells. As feeder cells for promoting sphere formation, any adherent cells can be used, but preferably feeder cells corresponding to various cells are desirable. Although not limited, for example, when forming a sphere of cells derived from liver or cartilage, examples of the feeder cells include COS-1 cells and vascular endothelial cells as a suitable cell type.
Additionally, culture compositions containing the nanofibers of the present invention can also be used to form spheres. At that time, the nanofibers may be added to the medium used in forming the spheres so that the concentration of the nanofibers becomes a concentration that enables suspension culture (preferably suspension stationary culture) of cells. For example, the concentration of the nanofibers is usually 0.0001% to 1.0% (weight / volume), for example 0.0005% to 1.0% (weight / volume), preferably 0.001% to 0. It is preferable that 3% (weight / volume), more preferably 0.005% to 0.1% (weight / volume), and still more preferably 0.01% to 0.05% (weight / volume). The fibers may be added to the medium used for sphere formation. The spheres are prepared by uniformly dispersing the target cells in the medium containing the nanofibers, and incubating the cells for 3 to 10 days. The spheres prepared here can be recovered by centrifugation or filtration. For example, the gravitational acceleration (G) at the time of centrifugation is 100 to 400 G, and the size of the pore of the filter used at the time of filtration is 10 μm to 100 μm, but is not limited thereto. In addition, spheres cultured by magnetic force can be recovered using magnetic fine particles coated on the surface with an antibody that specifically binds to a target cell. Examples of such magnetic fine particles include Dynabeads (manufactured by Veritas), MACS microbeads (manufactured by Milteny Biotech), BioMag (manufactured by Technochemical), and the like.

The size of the spheres varies depending on the cell type and culture period, but is not particularly limited, but in the case of spheres or oval spheres, diameters of 20 μm to 1000 μm, preferably 40 μm to 500 μm, more preferably 50 μm to 300 μm Have.
Such spheres can retain their proliferation ability for a period of 10 days or more, preferably 13 days or more, more preferably 30 days or more, by continuing the static culture as they are, but furthermore, periodically during static culture. By performing mechanical division, or by further performing unicellularization treatment and aggregation, proliferative ability can be maintained substantially indefinitely.
The culture vessel used for the culture of the spheres is not particularly limited as long as it can generally culture animal cells, and, for example, a flask, a dish, petri dishes, a dish for tissue culture, a multidish, a microplate, a micro Well plates, multiplates, multiwell plates, chamber slides, petri dishes, tubes, trays, culture bags, roller bottles and the like can be mentioned.
The medium used for static culture of the spheres can contain a cell adhesion factor, examples of which include Matrigel, collagen gel, gelatin, poly-L-lysine, poly-D-lysine, laminin, fibronectin It can be mentioned. These cell adhesion factors can also be added in combination of 2 or more types. Furthermore, thickeners such as guar gum, tamarind gum, propylene glycol alginate, locust bean gum, gum arabic, tara gum, methylcellulose and the like can be further mixed with the medium used for culturing the spheres.
By using the medium composition containing the nanofibers of the present invention, it is possible to uniformly disperse in the culture solution without performing operations such as shaking. It can be cultured as spheres without loss. Spheres statically cultured by this method can be recovered by centrifugation or filtration after culture. At this time, after adding the liquid medium used, centrifugation or filtration may be performed. For example, the gravitational acceleration (G) at the time of centrifugation is 100 to 400 G, and the size of the pore of the filter used at the time of filtration is 10 μm to 100 μm, but is not limited thereto. In addition, spheres cultured by magnetic force can be recovered using magnetic fine particles coated on the surface with an antibody that specifically binds to a target cell. Examples of such magnetic fine particles include Dynabeads (manufactured by Veritas), MACS microbeads (manufactured by Milteny Biotech), BioMag (manufactured by Technochemical), and the like. The recovered spheres can be dispersed as single cells by further processing using various chelating agents, heat, filters, enzymes and the like.

As a method for statically culturing plant-derived cells and / or tissues, calli, which is a non-differentiated plant cell mass, can be cultured. Callus can be induced by known methods for the plant species used. For example, 70% alcohol or 1% sodium hypochlorite, if necessary, on the surface of a part of tissue of a differentiated plant (eg, root, stem, leaf section, seed, growth point, embryo, pollen, etc.) After sterilization using a solution or the like, a tissue piece of appropriate size (for example, a root section of about 1 to about 5 mm square) is cut out using a scalpel or the like, and the tissue piece is subjected to aseptic operation using a clean bench or the like. Is inoculated into a previously sterilized callus induction medium and aseptically cultured under appropriate conditions. The callus derived here may be immediately subjected to liquid culture for mass growth, or can be maintained as a seed strain by subculturing in a subculture medium. The subculture may be performed using either a liquid medium or a solid medium.
The amount of plant cell mass inoculated when starting static culture using the medium composition of the present invention is the growth rate of the target cells, culture mode (batch culture, fed-batch culture, continuous culture, etc.), culture For example, when cultivating a plant cell mass such as callus, the wet weight of the cell mass relative to the medium composition of the present invention is 4 to 8 (weight / volume (w / v))%, although it varies depending on the period etc. The medium composition of the present invention is preferably inoculated at 5 to 7 (w / v)%. The particle size of the plant cell mass during culture is 3 mm to 40 mm, preferably 3 mm to 20 mm, more preferably 5 mm to 15 mm. Here, "particle size" means, for example, the diameter of a plant cell cluster if it is spherical, mean the major axis if it is elliptical spherical, and the maximum length that can be taken in other shapes as well. Do.

The temperature at which cells and / or tissues are cultured is usually 25 to 39 ° C., preferably 33 to 39 ° C. in the case of animal cells. The CO ₂ concentration is usually 4 to 10% by volume, preferably 4 to 6% by volume, in the culture atmosphere. The culture period is usually 3 to 35 days, but may be freely set according to the purpose of culture. The culture temperature of the plant cells is usually 20 to 30 ° C., and if light is required, the culture may be performed under an illumination condition of an illumination intensity of 2000 to 8000 lux. The culture period is usually 3 to 70 days, but may be freely set according to the purpose of culture.

  When culturing cells and / or tissues by the method of the present invention, cells and / or tissues separately prepared for the culture composition of the present invention may be added and mixed so as to be uniformly dispersed. The mixing method in that case is not particularly limited, and examples thereof include manual mixing such as pipetting, and mixing using an apparatus such as a stirrer, a vortex mixer, a microplate mixer, and a shaker. After mixing, the culture solution may be allowed to stand still, and the culture solution may be rotated, shaken or stirred as necessary. The number of rotations and the frequency may be appropriately set according to the purpose of those skilled in the art. In addition, when it is necessary to replace the medium composition during the period of stationary culture, after separating the cells and / or tissues from the medium composition by centrifugation or filtration, a new medium composition is used as a cell. And / or may be added to the tissue. Alternatively, cells and / or tissues may be appropriately concentrated by centrifugation or filtration, and then a new medium composition may be added to the concentrate. For example, the gravitational acceleration (G) at the time of centrifugation is 100 to 400 G, and the size of the pore of the filter used at the time of filtration is 10 μm to 100 μm, but is not limited thereto. Moreover, cells and / or tissues cultured by magnetic force can be separated using magnetic fine particles coated on the surface with an antibody that specifically binds to a target cell. Examples of such magnetic fine particles include Dynabeads (manufactured by Veritas), MACS microbeads (manufactured by Milteny Biotech), BioMag (manufactured by Technochemical), and the like. The exchange of these medium compositions can also be performed by a bioreactor or an automatic culture apparatus that can be performed under mechanical control and under a closed environment.

[Method for preserving or transporting cells or tissues]
The present invention also provides a storage method and a transport method for storing cells or tissues, using the medium composition of the present invention. In the storage or transport method of the present invention, cells or tissues can be stored or transported in a floating state (preferably, in a floating state) by using the medium composition of the present invention.

  Examples of cells and tissues to be stored or transported include those described above as cells and tissues that can be used for culture using the medium composition of the present invention.

  The medium composition of the present invention used for storage or transport may contain, in addition to the above-mentioned composition, various components having a cell survival effect at the time of storage of cells or tissues in a non-freezing state. As the components, saccharides (but excluding polysaccharides) (eg, monosaccharides, disaccharides), antioxidants (eg, SOD, vitamin E or glutathione), hydrophilic polymers (eg, polyvinyl pyrrolidone), chelating agents Examples include (eg, EDTA), sugar alcohols (eg, mannitol, sorbitol), glycerol and the like.

  In the storage or transport method of the present invention, desired cells or tissues are dispersed in the medium composition of the present invention and placed in a sealable container. Examples of the container include, but are not limited to, a flask, a plastic bag, a Teflon (registered trademark) bag, a tube, and a culture bag. A container containing a dispersion of cells or tissue in the medium composition of the present invention is preferably sealed to avoid leakage of contents and contamination of external bacteria during storage or transport. .

  The temperature during storage or transport is not particularly limited as long as cell or tissue survival is maintained, but is usually 37 ° C. or less. Lower temperatures may avoid loss of cell or tissue viability during storage or transport, but generally above the melting point of the media composition of the invention so that the cells or tissue do not freeze. Store or transport at temperature. Therefore, the temperature during storage or transport is usually maintained at -5 to 42 ° C, preferably 1 to 37 ° C, more preferably 4 to 32 ° C, still more preferably 18 to 30 ° C.

  The temperature during storage or transport is a temperature at which the medium composition of the present invention enables cell or tissue suspension, in order to allow storage or transport of cells or tissue in suspension state. Is preferred. The temperature at which cells or tissues can be suspended and set can be appropriately set according to the type of raw material constituting the nanofibers.

  In one aspect, carrageenan (preferably, κ-carrageenan) is used as a raw material constituting nanofibers contained in the medium composition of the present invention, which is used in the storage or transport method of the present invention. The medium composition of the present invention containing nanofibers composed of carrageenan has a floating action at 25 ° C. or less, but loses the floating action at 37 ° C., so at 25 ° C. or less (preferably 0 to 25 ° C.) The desired cells or tissues are stored or transported in a floating state, and after storage or transportation is completed, the temperature is raised to 37 ° C. or higher (eg, 37 to 40 ° C., preferably 37 ° C.) to suspend The cells or tissues can be easily recovered by precipitating the cells or tissues.

  The period of storage or transport is not particularly limited as long as cells or tissues can be maintained in the medium composition of the present invention, but usually 1 hour or more, 10 days or less, preferably 1 to 8 days, Preferably, it is 1 to 3 days. It is preferred that the cells or tissues be kept suspended in the medium composition of the present invention during the storage or transport period.

  By using the storage or transport method of the present invention, cells and tissues can be held in a suspended state, so damage to cells or tissues due to detachment from the plate due to vibration during transport or aggregation of cells or tissues contacted by sedimentation To preserve and transport cells and tissues while maintaining their original function.

  Hereinafter, the present invention will be described in more detail by specifically describing examples of the medium composition of the present invention. The materials, amounts used, proportions, treatment contents and treatment procedures shown in the following examples can be appropriately changed without departing from the spirit of the present invention. Therefore, the scope of the present invention should not be construed as the specific examples shown below.

Reference Example 1 Viscosity measurement and cell floating test of medium containing high temperature heat-treated deacylated gellan gum
Preparation of deacylated gellan gum-containing medium and viscosity measurement After suspending deacylated gellan gum (KELCOGEL CG-LA, manufactured by Tri Crystal Co., Ltd.) to 0.4% (w / v) in pure water, The mixture was heated and stirred at 90 ° C. for dissolution. The aqueous solution was allowed to cool to room temperature while stirring, and autoclaved at 121 ° C. for 20 minutes. In a 300 mL tall beaker, 50 mL of double concentration DMEM / F-12 medium (manufactured by Aldrich) and 47.5 mL of sterile water are added, and 2.5 mL of deacylated gellan gum aqueous solution is added while stirring with a homomixer (3000 rpm) at room temperature Then, stirring was continued for 1 minute as it was to prepare a medium composition with a final concentration of 0.01% deacylated gellan gum. Similarly, a medium composition was prepared by adding a deacylated gellan gum aqueous solution to a final concentration of 0.02, 0.03, 0.05% (w / v). The viscosity of this medium composition was measured at 37 ° C. for 5 minutes using an E-type viscometer (manufactured by Toki Sangyo Co., Ltd., Viscometer TVE-22L, standard rotor 1 ° 34 ′ × R24) at a rotational speed of 100 rpm. .

Cell suspension test of deacylated gellan gum-containing medium Human cervical cancer cell line HeLa (DS Pharma Biomedical Co., Ltd.), 250000 cells / well in EMEM medium (WAKO) containing 10% (v / v) fetal bovine serum The suspension was suspended to be mL, and 10 mL of this suspension was seeded on EZ SPHERE (manufactured by Asahi Glass Co., Ltd.), and then cultured for 3 days in a CO ₂ incubator (5% CO ₂ ). 10 mL of the suspension of spheres (diameter 100 to 200 μm) obtained here was centrifuged (200 G, 5 minutes) to precipitate the spheres, and 1.0 mL of sphere suspension was prepared by removing the supernatant. Subsequently, 1.0 mL each of the deacylated gellan gum-containing medium prepared above was placed in a 1.5 mL eppendorf tube, and 10 μL of HeLa cell sphere suspension was further added. The cell mass was dispersed by tapping, incubated at 37 ° C., and the dispersion state of the cells after 1 hour was visually observed.

Reference comparative example Preparation of methylcellulose and collagen-containing medium
Preparation of Methylcellulose- Containing Medium 100 mL of DMEM / F-12 medium (manufactured by Aldrich) was placed in a 200 mL eggplant flask, and 0.1 g of methylcellulose (M0387, manufactured by Aldrich) was added. Stir while cooling with an ice bath to dissolve methylcellulose. A medium composition was prepared by adding a methylcellulose aqueous solution to a final concentration of 0.1, 0.3, 0.6, 1.0% (w / v) using this solution.
Preparation of Collagen-Containing Medium 1 mL of 10 × DMEM / F-12 medium (Aldrich) in 6.5 mL of 0.3% cell matrix type I (Nitta Gelatin Co., Ltd.), buffer for reconstitution (new) 1 mL of Ta gelatin Co., Ltd. and 1.5 mL of pure water were added, and 0.2% collagen-containing medium was prepared while stirring in ice. Similarly, a culture medium composition was prepared in which collagen was added to a final concentration of 0.01, 0.05, 0.1, 0.2% (w / v).
The suspension test and viscosity measurement of HeLa cell spheres were carried out for the medium composition prepared above as well as the medium containing the deacylated gellan gum. However, the viscosity of 1.0% (w / v) methylcellulose was measured at 50 rpm from the measurement range of the device.

Reference Test Example In the following reference test example, the concentration (%) of CO ₂ in the CO ₂ incubator is indicated by the volume% of CO _{2 in} the atmosphere. Further, PBS means phosphate buffered saline (manufactured by Sigma Aldrich Japan), and FBS means fetal calf serum (manufactured by Biological Industries). Also, (w / v) represents weight per volume.

Reference test example 1: Cell proliferation test when single cells are dispersed Ultra-pure so that deacylated gellan gum (KELCOGEL CG-LA, manufactured by Three Crystal Co., Ltd.) is 0.3% (w / v) After suspending in water (Milli-Q water), it was dissolved by stirring with heating at 90 ° C., and the aqueous solution was autoclaved at 121 ° C. for 20 minutes. Using this solution, deacylation with a final concentration of 0.015% (w / v) in IMDM medium (Gibco) containing 10% (v / v) fetal bovine serum and 10 ng / mL thrombopoietin (WAKO) A medium composition to which a gellan gum had been added was prepared. Subsequently, the human leukemia cell line UT7 / TPO is seeded on the medium composition to which the above-mentioned deacylated gellan gum is added to 20000 cells / mL, and then 1 well of a 6-well flat bottom microplate (manufactured by Corning) The aliquots were aliquoted to 5 mL per well. Similarly, a human cervical cancer cell line HeLa (manufactured by DS Pharma Biomedical Co., Ltd.) was added to EMEM medium (manufactured by WAKO) containing 10% (v / v) fetal bovine serum (manufactured by WAKO) to 20000 cells / mL. After seeding on a medium composition to which 015% (w / v) of deacylated gellan gum (KELCOGEL CG-LA, manufactured by Tri Crystal Co., Ltd.) has been added, 1 well of a 6-well flat bottom microplate (manufactured by Corning) is used. It was aliquoted to 5 mL per volume. These cell suspensions were cultured still in a CO ₂ incubator (5% CO ₂ ) for 3 days. Thereafter, a portion of the culture solution was recovered, and after adding equal amounts of trypan blue staining solution (manufactured by Invitrogen), the number of living cells was measured with a hemacytometer (manufactured by Elma Sales Co., Ltd.).

  As a result, UT7 / TPO cells and HeLa cells can be uniformly cultured in a floating state by using the above-mentioned medium composition, and it was confirmed that they proliferate in the medium composition. The cell numbers of UT7 / TPO cells and HeLa cells after 3 days of suspension static culture are shown in Table 4.

Reference Test Example 2: Cell proliferation test when culturing cell line-derived spheres DMEM medium (WAKO Co., Ltd.) containing 10% (v / v) fetal bovine serum of human hepatoma cell line HepG2 (DS Pharma Biomedical Co., Ltd.) ) Was suspended to 250000 cells / mL, and 10 mL of the suspension was seeded on EZ SPHERE (manufactured by Asahi Glass Co., Ltd.), and then cultured for 7 days in a CO ₂ incubator (5% CO ₂ ). Similarly, human cervical cancer cell line HeLa (manufactured by DS Pharma Biomedical Co., Ltd.) is suspended in EMEM medium (manufactured by WAKO) containing 10% (v / v) fetal bovine serum to a concentration of 250000 cells / mL. After 10 mL of the suspension was seeded on EZ SPHERE (manufactured by Asahi Glass Co., Ltd.), the suspension was cultured in a CO ₂ incubator (5% CO ₂ ) for 7 days. A suspension of 2.5 mL of the suspension (diameter: 100 to 200 μm) of each cell line thus obtained was centrifuged (200 G, 5 minutes) to precipitate the sphere and remove the supernatant. Subsequently, 10 mL of the above medium was added to the spheres (about 800) and suspended, and then transferred to a flat bottom tube (manufactured by BM Instruments Co., Ltd.). Similarly, a suspension of spheres is prepared using a medium composition obtained by adding 0.015% (w / v) of deacylated gellan gum (KELCOGEL CG-LA, manufactured by Three Crystal Co., Ltd.) to the above medium, It transferred to the flat bottom tube (made by BM apparatus company). In addition, the medium composition to which 0.015% (w / v) of deacylated gellan gum was added was 0.3% (w / v) of deacylated gellan gum (KELCOGEL CG-LA, manufactured by Three Crystal Co., Ltd.) first. The suspension is suspended in ultrapure water (Milli-Q water) so as to become), dissolved by stirring while heating at 90 ° C, and this aqueous solution is autoclaved at 121 ° C for 20 minutes, and then diluted to 1/20. And 10% (v / v) fetal bovine serum by adding to DMEM medium.

After static culture of the above sphere suspension in a CO ₂ incubator (5% CO ₂ ) at 37 ° C. for 3 days, a 2-fold volume of medium is added and centrifugation (200 G, 5 minutes) The spheres were allowed to settle and the supernatant was removed. Here, a part of the sphere was separated, and the shape was observed with an optical microscope (CK30-F100 manufactured by OLYMPUS, Inc.). Subsequently, the collected spheres were washed once with 10 mL of PBS, and 1 mL of a trypsin-EDTA (ethylenediaminetetraacetic acid) solution (manufactured by WAKO) was added and incubated at 37 ° C. for 5 minutes. After 9 mL of the above-mentioned culture medium was added, cells were collected by centrifugation (200 G, 5 minutes). An equal amount of trypan blue staining solution (manufactured by Invitrogen) is added to a part of 2 mL of the cell suspension obtained here, and then living cells and death are performed with a hemacytometer (manufactured by Elma Sales Co., Ltd.) The number of cells was measured.

As a result, it was possible to culture the spheres of HepG2 cells and HeLa cells in a suspended state by using the above-mentioned medium composition, and it was confirmed that the cells proliferate efficiently by the medium composition. Moreover, it was confirmed that the medium composition has a smaller proportion of dead cells when cells are allowed to grow as compared to the existing medium, and that the effect of promoting cell growth is excellent. At this time, the spheres cultured in the existing medium were sedimented on the bottom of the culture vessel. Furthermore, when the shape of the cultured spheres was observed with a light microscope, association of the spheres was not observed in the medium composition, whereas association of the spheres was observed in the existing medium.
Table 5 shows the relative numbers of HepG2 cells and HeLa cells when the number of cells is 1 when cultured in a medium without deacylated gellan gum. The relative dead cell rate is shown in Table 6 when the dead cell rate (the number of dead cells / the number of viable cells) when cultured in a medium not containing deacylated gellan gum is 1. Also, the suspended state when HepG2 cells and HeLa cells spheres are cultured with the medium composition is shown in FIGS. 1 and 2, respectively. Furthermore, the shape of the cultured HeLa cell spheres is shown in FIG.

Reference Test Example 3 Cell Proliferation Test When Cultured Cell Line Adhered on Microcarrier A microcarrier Cytodex (registered trademark) 1 (manufactured by GE Healthcare Life Sciences) was suspended in PBS so as to be 0.02 g / mL. After being cloudy and standing overnight, the supernatant was discarded and the microcarrier was washed twice with fresh PBS. Thereafter, the cells were suspended again in PBS to 0.02 g / mL, and autoclaved at 121 ° C. for 20 minutes. Subsequently, the microcarriers are washed twice with 70% ethanol and three times with PBS, and then 0.02 g / mL with DMEM medium (WAKO) containing 10% (v / v) fetal bovine serum. Suspended to become Using this microcarrier suspension, prepare 20 mL of DMEM medium (containing 10% (v / v) fetal bovine serum) containing 120 mg of Cytodex (registered trademark) 1 and 4,000,000 HepG2 cells, and suspend this cell suspension. The solution was cultured in a beaker previously treated with a silicone coating agent (manufactured by Asahi Techno Glass Co., Ltd.) at 37 ° C. for 6 hours while stirring (100 rpm) with a stirrer. Here, it was confirmed with a microscope that HepG2 cells were adhered to the microcarrier. Subsequently, the microcarriers to which cells were attached were washed twice with DMEM medium containing 10% (v / v) fetal bovine serum and suspended in 3 mL of the same medium.

  Media composition prepared by adding 20 mL of DMEM medium containing 10% (v / v) fetal bovine serum or 0.015% (w / v) deacylated gellan gum (KELCOGEL CG-LA, manufactured by Three Crystal Co., Ltd.) to this medium 300 μL of the above-mentioned microcarrier suspension was added to each of the above, and cultured at 37 ° C. for 3 days. At this time, the culture solution containing no deacylated gellan gum was cultured while stirring (100 rpm) with a stirrer. After culture, the adhesion state of the cells on the microcarriers was confirmed with a microscope, and the microcarriers were precipitated by centrifugation (200 G, 5 minutes). After washing this microcarrier with 10 mL of PBS, 1 mL of a trypsin-EDTA (ethylenediaminetetraacetic acid) solution (manufactured by WAKO) was added, and the mixture was incubated at 37 ° C. for 5 minutes. Furthermore, 9 mL of DMEM medium containing 10% (v / v) fetal bovine serum was added, and then the microcarriers were removed using a cell strainer with a mesh size of 70 μm (manufactured by BD Falcon). Cells were recovered from the filtrate obtained here by centrifugation (200 G, 5 minutes). The cells are suspended in 500 μL of culture medium, and after adding an equal amount of trypan blue staining solution (manufactured by Invitrogen) to a portion of the suspension, the number of living cells is measured with a hemocytometer (manufactured by Elma Sales Co., Ltd.) Was measured. As a result, the medium without deacylated gellan gum contained 123,000 cells, while the medium containing deacylated gellan gum contained 1,320,000 cells. As described above, it was confirmed that the medium composition containing the structure of the specific compound is more effective in promoting cell growth as compared to the existing medium, even when cell culture is performed using a microcarrier. . The adhesion state of HepG2 cells when microcarrier culture is performed for 3 days using a medium composition containing the structure of the specific compound is shown in FIG.

Reference Test Example 4: Cell floating test using cell line-derived spheres Xanthan gum (KELTROL CG, manufactured by Tri Crystal Co., Ltd.) in ultrapure water (Milli-Q water) to a concentration of 1% (w / v) After suspension, it was dissolved by stirring with heating at 90.degree. This aqueous solution was used to prepare a DMEM / F-12 medium composition having a final concentration of 0.1, 0.15, 0.2% (w / v) for xanthan gum. In addition, 0.2% (w / v) −-carrageenan (GENUGEL WR-80-J, manufactured by Tri Crystal Co., Ltd.) and 0.2% (w / v) locust bean gum (GENUGUM RL-200-J) ), And the aqueous solution containing 0.03, 0.04, 0.05% (w / v) of κ-carrageenan and locust. A DMEM / F-12 medium (manufactured by Sigma) composition containing bean gum was prepared.

  Spheres of HeLa cells were prepared using the same method as in Reference Test Example 2, and after adding several tens of spheres to 1 mL of the above-prepared medium, each was allowed to stand at 37 ° C. for 1 hour. The floating state of was visually observed. As a result, it was confirmed that the spheres of HeLa cells were maintained in a suspended state in all of the above medium compositions. Furthermore, after adding an equal volume of culture medium to the cell suspension, it was confirmed that the spheres of HeLa cells were sedimented and recovered by centrifugation (300 to 400 G, 5 minutes). The suspended state when HeLa cell spheres are cultured with the medium composition is shown in FIG. Moreover, the viscosity measured by the method similar to analysis example 1 is shown in Tables 7 and 8.

Reference Test Example 5 Cell Suspension Test Using Filtered Media Composition Using the same method as Reference Test Example 2, 0.015% deacylated gellan gum (KELCOGEL CG-LA, manufactured by Sanyo Co., Ltd.) was used. A containing DMEM / F-12 medium composition was prepared. Subsequently, 1 mL of the present medium composition is subjected to 70 μm, 40 μm filter (manufactured by BD Falcon), 30 μm, 20 μm filter (manufactured by As One), 10 μm filter (manufactured by Partec), 5 μm, 1.2 μm, 0.45 μm, It filtered using a 0.2 micrometer filter (made by Sartorius Stedim Japan), respectively. After adding several dozens of HepG2 cell spheres prepared using the same method as in Reference Test Example 2 to the above-mentioned filtrate, the cells are allowed to stand at 37 ° C. for 1 hour to suspend sphere cells Were visually observed. As a result, it was confirmed that the spheres of HepG2 cells were maintained in the suspended state in the medium composition that permeated the filter of 10 μm or more, but precipitated in the medium composition that permeated the filter of 5 μm or less. Furthermore, the spheres of HepG2 cells in suspension here are sedimented by centrifugation at 300 G for 5 minutes at room temperature, or by adding an equal volume of culture medium followed by centrifugation at 200 G for 5 minutes at room temperature. Confirmed that it could be recovered.

Reference Test Example 6: Sphere formation test 0.01% deacylated gellan gum (KELCOGEL CG-LA, manufactured by Three Crystal Co., Ltd.) and 10% (v / v) fetal bovine using the same method as in Reference Test Example 2. A composition of EMEM medium (manufactured by WAKO) containing serum was prepared. Subsequently, HeLa cells were added to a concentration of 1000 cells / mL and then aliquoted into a 24-well plate (manufactured by Corning). This plate was suspended and cultured at 37 ° C. for 9 days, and then formation of spheres was confirmed by a microscope. Furthermore, sphere cells are sedimented by centrifugation at 300 G for 5 minutes, washed once with 5 mL of PBS, 100 μL of trypsin-EDTA (ethylenediaminetetraacetic acid) solution (WAKO) is added, and the solution is kept at 37 ° C. for 5 minutes. I kept warm. To 100 μL of the cell suspension obtained here, 100 μL of EMEM medium containing 10% (v / v) fetal bovine serum was added, and a portion of the cell suspension was subjected to trypan blue staining (in vitro After adding the same amount of JEN Co., Ltd.), the number of living cells was measured with a hemacytometer (manufactured by Elma Co., Ltd.). As a result, it was confirmed that HeLa cells were increased to 170000 cells / mL. Spheres of HeLa cells formed with the medium composition are shown in FIG.

Reference Test Example 7: Optical microscope observation of the structure After suspending the deacylated gellan gum (KELCOGEL CG-LA, manufactured by Tri Crystal Co., Ltd.) to 0.4% (w / v) in pure water, The mixture was heated and stirred at 90 ° C. for dissolution. Put 95 mL of double concentration DMEM / F-12 medium (manufactured by Aldrich) in a 300 mL tall beaker, add 5 mL of deacylated gellan gum aqueous solution while stirring with a magnetic stirrer at room temperature, and continue stirring for 5 minutes as it is A final medium composition with a final concentration of 0.02% deacylated gellan gum was prepared. Furthermore, the medium composition was stirred for 5 minutes with a homomixer (3000 rpm). The prepared medium composition was observed with a light microscope (KEYENCE, BIOREVO BZ-9000). The observed structure is shown in FIG.

Reference Test Example 8 Preparation by Mixed Heating of Powdered Medium and DAG 200 mg of deacylated gellan gum (KELCOGEL CG-LA, manufactured by Three Crystal Co., Ltd.) 20 mg and 1.58 g of DMEM / F-12 medium (manufactured by Life Technologies) The flask was placed in an Erlenmeyer flask and 100 mL of pure water was poured. The autoclave was sterilized at 121 ° C. for 20 minutes to prepare a DMEM / F-12 medium composition having a deacylated gellan gum concentration of 0.02%. To the prepared medium, dextran beads Cytodex 1 (Size 200 μm, manufactured by GE Healthcare Life Sciences) were added, and the dispersion state of the beads was visually confirmed. The floating dispersion state was evaluated as ○, the partial sedimentation / dispersion state was Δ, and the sedimentation state was evaluated as x. The results are shown in Table 9.

Reference Test Example 9 Preparation of Medium Composition Mixed with Polysaccharide After xanthan gum (KELTROL CG, manufactured by Tri Crystal Co., Ltd.) is suspended in pure water to a concentration of 0.5% (w / v), It melt | dissolved by stirring, heating at 90 degreeC. Similarly, sodium alginate (Duck alginic acid NSPM, manufactured by Hood Chemifa), locust bean gum (GENUGUM RL-200-J, manufactured by Sanyo Co., Ltd.), κ-carrageenan (GENUGEL WR-80-J, manufactured by Sanyo Co., Ltd.) A 0.5% (w / v) aqueous solution was prepared for Daiyutan gum (KELCO CRETE DG-F, manufactured by Three Crystal Co., Ltd.).
This aqueous solution was mixed with a 0.2 or 0.1% (w / v) deacylated gellan gum solution and a 10-fold concentration DMEM / F-12 medium, and heated at 80 ° C. for 30 minutes. After cooling to room temperature, a 7.5% aqueous sodium bicarbonate solution is added to a final concentration of 0.01, 0.02% (w / v) of a deacylated gellan gum and a final concentration of 0.1, 0.2, A DMEM / F-12 medium composition was prepared containing 0.3, 0.4% (w / v) other polysaccharides. Also, after preparing a medium containing deacylated gellan gum as described above, a powder of methylcellulose (cP400, manufactured by WAKO Co., Ltd.) was added. Stir in ice bath to dissolve methylcellulose, final concentration 0.01, 0.02% (w / v) deacylated gellan gum and final concentration 0.1, 0.2, 0.3, 0.4 A DMEM / F-12 medium composition containing% (w / v) other methylcellulose was prepared.

  Polystyrene beads (Size 500-600 μm, manufactured by Polysciences Inc.) were added to the medium prepared above, and the dispersion state of the beads was visually confirmed. The floating dispersion state was evaluated as ○, the partial sedimentation / dispersion state Δ, and the sedimentation state as x. The results are shown in Table 10.

Reference test example 10: Viscosity measurement of medium composition mixed with polysaccharides Deacylation with a final concentration of 0.005 and 0.01% (w / v) in the same manner as in the polysaccharide mixed system of Reference Test Example 9 DMEM / F-12 medium was prepared containing gellan gum and other polysaccharides. The final concentrations of polysaccharides are xanthan gum, sodium alginate, locust bean gum 0.1% (w / v), methylcellulose 0.2% (w / v), κ-carrageenan and diutatan gum 0.05% (w) It was prepared to be / v). The condition of each medium composition and the viscosity measured by the same method as that of Analysis Example 1 are shown in Tables 11-16.

Reference Test Example 1: Preparation of Medium Composition Having Different Concentrations of Metal Ions Using DMEM / F-12 (D9785, Aldrich) without calcium chloride, magnesium sulfate and magnesium chloride, the method of Reference Test Example 8 Similarly, a DMEM / F-12 medium composition containing 0.02% (w / v) of deacylated gellan gum was prepared. Moreover, the DMEM / F-12 culture medium composition which added calcium chloride or magnesium sulfate and magnesium chloride so that a final concentration might become a defined quantity of a DMEM / F-12 culture medium was prepared. From the defined composition of the DMEM / F-12 medium, the final concentrations of each were 0.116 g / L of calcium chloride, 0.049 g / L of magnesium sulfate, and 0.061 g / L of magnesium chloride.
Dextran beads Cytodex 1 (manufactured by GE Healthcare Life Sciences) were added to the prepared medium composition, and dispersion of the beads was visually confirmed after 2 days. The floating dispersion state was evaluated as ○, the partial sedimentation / dispersion state was Δ, and the sedimentation state was evaluated as x. The results are shown in Table 17.

Reference Test Example 12 Preparation of Medium Composition Post-added with Divalent Metal Cation 0.1% (w / v) Deacylated Gellan Gum Solution and DMEM / F-12 Medium (Calcium Chloride, Magnesium Sulfate, 5-fold Concentration) A salt solution was prepared by dissolving magnesium chloride free, D9785 (manufactured by Aldrich), calcium chloride 1167 mg, magnesium sulfate 489 mg, and magnesium chloride 287 mg in 300 mL of pure water. A deacylated gellan gum aqueous solution and pure water were placed in a 200-mL tall beaker, and the solution was stirred at 200 rpm using an electric stirrer blade. Solution A in which the medium solution and water were mixed was added, and the solution was stirred for 10 minutes as it was. Then, a salt solution was added, and further 1.6 mL of 7.5% aqueous sodium hydrogen carbonate solution was added to prepare a DMEM / F-12 medium composition containing a final concentration of 0.02% deacylated gellan gum. The mixing amount of each liquid is shown in the table. After 4 hours of preparation, the dispersion evaluation of polystyrene beads and Cytodex 1 was performed on 6 medium compositions. The results are shown in Tables 18 and 19.

Reference Test Example 13 Preparation of Various Medium Composition A 0.1% (w / v) deacylated gellan gum solution and a high concentration medium solution were prepared. As the high concentration medium solution, 10 times concentration MEM (M0268, Aldrich), RPMI-1640 (R6504, Aldrich) and 5 times concentration DMEM (high pressure sterilization compatible medium, Nissui) were prepared. A 0.1% (w / v) deacylated gellan gum solution, each high concentration medium, and pure water for concentration adjustment were mixed, and heated at 80 ° C. for 30 minutes. After cooling to room temperature, 7.5% aqueous sodium hydrogen carbonate solution is added to the medium composition containing final concentrations of 0.01, 0.02, and 0.03% (w / v) of deacylated gellan gum, respectively. Prepared.
With respect to the prepared six medium compositions, the floating dispersion state of polystyrene beads and dextran beads Cytodex 1 was evaluated as 沈降, partial sedimentation / dispersion state Δ, and the sedimentation state as x. The results are shown in Tables 20 and 21.

Reference Test Example 14 Measurement of Particle Size Distribution of Media Composition Containing Deacylated Gellan Gum According to Reference Example 1, a DMEM / F-12 media composition containing 0.038% (w / v) of deacylated gellan gum is prepared did. The medium was prepared by stirring at 3000 rpm and 6000 rpm for 1 minute using a homomixer. The particle size distribution of the present medium composition was measured using Beckman Coulter Co., Ltd. product Multisizer 4 (precise particle size distribution measuring device by Coulter principle), and the median diameter (d50) of the volume-based particle size distribution was determined. The results are shown in Table 22.

Reference Test Example 15 1 g of deacylated gellan gum and 40 mL of pure water were weighed into a 100 mL test tube of a phosphated glass of deacylated gellan gum, and heated at 100 ° C. for 30 minutes to prepare a suspension. To this suspension, 1 g of an aqueous phosphoric acid solution (85%) was added and heated to reflux for 5 hours. Then, while stirring for 12 hours, the white suspension obtained by cooling to room temperature was poured into 99% ethanol (500 mL). The resulting fluffy white solid was collected by filtration and dried to obtain a pale brown solid (0.4 g) as a phosphate of deacylated gellan gum. The introduction of the phosphate group was confirmed by Fourier transform infrared spectroscopy (IR-Prestage 21 manufactured by Shimadzu Corporation) (1700 cm -1; P-OH, 1296 cm -1, 1265 cm -1; P = O ). A light brown solid is decomposed by microwave thermal decomposition equipment (ETHOS TC, manufactured by Milestone General), and then contains phosphorus atoms by inductively coupled plasma emission spectrometry (ICP-OES) (SPS 5520, manufactured by SII Nanotechnology Inc.) As a result of measuring the rate, it was 3.5 wt% (n = 2).

Reference Test Example 16 Preparation of Medium Composition Containing Phosphorylated Deacylated Gellan Gum An arbitrary amount of phosphorylated deacylated gellan gum (30 mg) and 1.56 g of DMEM / F-12 medium (manufactured by Life Technologies) were prepared. The mixture was placed in a 200 mL Erlenmeyer flask and 100 mL of pure water was poured. The autoclave was sterilized at 121 ° C. for 20 minutes to prepare a DMEM / F-12 medium composition having a deacylated gellan gum concentration of 0.03%. To the prepared medium, dextran beads Cytodex 1 (manufactured by GE Healthcare Biosciences) were added, and the dispersion state of the beads was visually confirmed. Dispersion of the beads was observed at a concentration of 0.03% (w / v) phosphorylated deacylated gellan gum.

Reference test example 17: Preparation of medium composition containing deacylated gellan gum The deacylated gellan gum aqueous solution and the medium solution are mixed by adding them in the proportions shown in the table below, and the deacylated gellan gum concentration is 0.02%. The dispersion state of polystyrene beads (Size 500-600 μm, manufactured by Polysciences Inc.) when the DMEM / F-12 medium composition was prepared was evaluated. The results are shown in Tables 23 and 24. By standing still for one day or more, styrene beads were dispersed under all conditions.

Reference Test Example 18 Preparation of Medium Composition Using Filter Deacylated gellan gum (KELCOGEL CG-LA, manufactured by Three Crystal Co., Ltd.) to a final concentration of 0.02 or 0.04% (w / v) The solution was suspended in ultrapure water (Milli-Q water) and dissolved by heating at 90.degree. C. for 30 minutes or at 121.degree. C. for 20 minutes. Furthermore, 100 mL of the aqueous solution was filtered through a polyethersulfone membrane filter (manufactured by Corning) having a pore size of 0.22 μm. Subsequently, the filtrate is mixed with a 2 to 4 times concentration DMEM / F-12 medium (manufactured by Sigma Aldrich), and shaken with a mild mixer (SI-24, manufactured by Taitec) for 1 hour, and the final A medium composition containing 0.01 or 0.015% (w / v) deacylated gellan gum was prepared respectively (for example, 0.02% (w / v) deacylated gellan gum aqueous solution and twice the concentration) 25 mL of DMEM / F-12 medium were mixed to prepare 50 mL of 0.01% (w / v) deacylated gellan gum media composition). Spheres of HepG2 cells were prepared using the same method as in Reference Test Example 2, and after adding several tens of spheres to 1 mL of the culture medium prepared above, each was allowed to stand at 37 ° C. for 1 hour and 1 The floating state of sphere cells after night was visually observed. As a result, it was confirmed that the spheres of HepG2 cells were maintained in a suspended state in all of the above medium compositions. Furthermore, after adding 2 volumes of medium, the cell suspension was centrifuged (500 G, 5 minutes) to precipitate the spheres of HepG2 cells, and it was confirmed in all medium compositions that cells could be recovered. . When the dispersion state of the sphere after one night was visually confirmed, the results obtained by evaluating the floating dispersion state as 沈降, partial sedimentation / dispersion state as Δ, and the sedimentation state as x are shown in Table 25.

Reference test example 19: Cell proliferation test when culturing cell line-derived spheres EMEM medium (WAKO Co., Ltd.) containing 10% (v / v) fetal bovine serum of human fetal kidney cell line HEK293 (made by DS Pharma Biomedical Co., Ltd.) And 25 mL / mL of this suspension, and 10 mL of this suspension was seeded on EZ SPHERE (manufactured by Asahi Glass Co., Ltd.), and then cultured for 2 days in a CO ₂ incubator (5% CO ₂ ). 10 mL of the suspension of the sphere (diameter 100 to 200 μm) of the HEK 293 cells obtained here was centrifuged (200 G, 5 minutes) to sediment the sphere and remove the supernatant, and then suspended in 1 mL. Subsequently, 10 mL of the above medium was added to 200 μL of the sphere suspension (cell number: approximately 200,000) and suspended, and then transferred to a flat bottom tube (manufactured by BM Instruments Co., Ltd.). Similarly, a suspension of spheres is prepared using a medium composition obtained by adding 0.015% (w / v) of deacylated gellan gum (KELCOGEL CG-LA, manufactured by Three Crystal Co., Ltd.) to the above medium, It transferred to the flat bottom tube (made by BM apparatus company). In addition, the medium composition to which 0.015% (w / v) of deacylated gellan gum was added was 0.3% (w / v) of deacylated gellan gum (KELCOGEL CG-LA, manufactured by Three Crystal Co., Ltd.) first. The suspension is suspended in ultrapure water (Milli-Q water) so as to become), dissolved by stirring while heating at 90 ° C, and this aqueous solution is autoclaved at 121 ° C for 20 minutes, and then diluted to 1/20. In EMEM medium containing 10% (v / v) fetal bovine serum.

After static culture of the above sphere suspension in a CO ₂ incubator (5% CO ₂ ) at 37 ° C. for 5 days, a 2-fold volume of culture medium is added and centrifugation (500 G, 5 minutes) is performed. The spheres were allowed to settle and the supernatant was removed. Subsequently, the collected spheres were washed once with 10 mL of PBS, and 1 mL of a trypsin-EDTA (ethylenediaminetetraacetic acid) solution (manufactured by WAKO) was added and incubated at 37 ° C. for 5 minutes. After 9 mL of the above-mentioned culture medium was added, cells were recovered by centrifugation (500 G, 5 minutes). An equal amount of trypan blue staining solution (manufactured by Invitrogen) is added to a part of 2 mL of the cell suspension obtained here, and then living cells and death are performed with a hemacytometer (manufactured by Elma Sales Co., Ltd.) The number of cells was measured. As a control, a medium composition not containing deacylated gellan gum was prepared, and the same experiment was performed.

As a result, it was possible to culture the spheres of HEK 293 cells in a suspended state by using the medium composition, and it was confirmed that the cells efficiently proliferated in the medium composition. Moreover, it was confirmed that the medium composition has a smaller proportion of dead cells when the cells are allowed to grow as compared with the medium composition not containing deacylated gellan gum, and the cell growth promoting effect is excellent. At this time, the spheres cultured in the existing medium were sedimented on the bottom of the culture vessel.
Table 26 shows the relative cell number when HEK 293 cells are cultured in a medium not containing deacylated gellan gum, and the cell number is 1. The relative dead cell rate is shown in Table 27 when the dead cell rate (the number of dead cells / the number of viable cells) is 1 when cultured in a medium not containing deacylated gellan gum.

Reference test example 20: Cell proliferation test when insect cells are cultured Ultrapure water (Milli) so that 0.3% (w / v) of deacylated gellan gum (KELCOGEL CG-LA, manufactured by Sanyo Co., Ltd.) is obtained. After suspending in -Q water, it was dissolved by stirring while heating at 90 ° C, and the aqueous solution was autoclaved at 121 ° C for 20 minutes. Using this solution, a medium composition was prepared by adding a final concentration of 0.015% (w / v) of deacylated gellan gum to Sf-900 (registered trademark) III SFM medium (Gibco). Subsequently, Sf9 cells (Gibco) derived from Spodoptera frugiperda are seeded on the medium composition to which the above-mentioned deacylated gellan gum is added so as to be 100,000 cells / mL, and then a 24-well flat bottom microplate (Corning) To 1 well per well. These cell suspensions were statically cultured at 25 ° C. for 5 days in an incubator. Thereafter, a portion of the culture solution was recovered, and after adding equal amounts of trypan blue staining solution (manufactured by Invitrogen), the number of living cells was measured with a hemacytometer (manufactured by Elma Sales Co., Ltd.). As a control, a medium composition not containing deacylated gellan gum was prepared, and the same experiment was performed.

  As a result, it was confirmed that Sf9 cells can be uniformly cultured in a floating state by using the medium composition, and proliferate in the medium composition. Furthermore, it was confirmed that the medium composition has an excellent cell growth promoting effect when cells are grown as compared to a medium composition not containing deacylated gellan gum. The cell numbers of Sf9 cells after 5 days of suspension static culture are shown in Table 28.

Reference test example 21: Cell proliferation test when CD34 positive cells are cultured Ultrapure water (KELCOGEL CG-LA, manufactured by Sanyo Co., Ltd.) to be 0.3% (w / v) ultrapure water (w / v) After suspending in Milli-Q water), the solution was dissolved by stirring with heating at 90 ° C., and the aqueous solution was autoclaved at 121 ° C. for 20 minutes. Using this solution, the final concentration of 0.015% (w / v) deacylated gellan gum in StemSpan SFEM medium (StemCell Technologies), 20 ng / mL thrombopoietin (WAKO) and 100 ng / mL stem cell factor ( A medium composition to which SCF (manufactured by WAKO) was added was prepared. Subsequently, CD34 positive cells derived from human umbilical cord blood (manufactured by Lonza) are seeded on the medium composition to which the above-mentioned deacylated gellan gum is added so as to be 10000 cells / mL, and then 24-well flat bottom microplate (Corning Inc.) ) To 1 mL per well. These cell suspensions were static cultured at 37 ° C. for 7 days in a CO ₂ incubator (5% CO ₂ ). Thereafter, a portion of the culture solution was recovered, and after adding equal amounts of trypan blue staining solution (manufactured by Invitrogen), the number of living cells was measured with a hemacytometer (manufactured by Elma Sales Co., Ltd.). In addition, all cells were sedimented by adding 3 volumes of medium to the remaining culture solution and performing centrifugation (500 G, 5 minutes). As a control, a medium composition not containing deacylated gellan gum was prepared, and the same experiment was performed.

  As a result, CD34 positive cells can be uniformly cultured in a suspended state by using the medium composition, and it was confirmed that they proliferate in the medium composition. Furthermore, it was confirmed that the medium composition has a cell growth promoting effect equal to or greater than that of the existing medium without deacylated gellan gum. In addition, it was confirmed that the cells were sedimented by centrifugation and the cells could be recovered. Table 29 shows the relative number of cells grown from CD34 positive cells after 7 days of floating stationary culture, where the number of cells when cultured in a medium not containing deacylated gellan gum is 1.

Reference Test Example 22 Sphere Formation Test 0.015% deacylated gellan gum (KELCOGEL CG-LA, manufactured by Tri Crystal Co., Ltd.) and 10% (v / v) fetal bovine using the same method as Reference Test Example 2. A composition of DMEM medium (manufactured by WAKO) containing serum was prepared. Subsequently, HepG2 cells were added to a cell concentration of 15000 cells / mL, and then 1 mL was dispensed into a 24-well plate (manufactured by Corning). After suspension and culture of this plate for 7 days at 37 ° C., formation of spheres was confirmed with a microscope. Furthermore, sphere cells are sedimented by centrifugation at 400 G for 5 minutes, washed once with 5 mL of PBS, 100 μL of a trypsin-EDTA (ethylenediaminetetraacetic acid) solution (WAKO) is added, and the solution is kept at 37 ° C. for 5 minutes. I kept warm. To 100 μL of the cell suspension obtained here, 100 μL of DMEM medium containing 10% (v / v) fetal bovine serum was added, and a portion of the cell suspension was subjected to trypan blue staining (in vitro After adding the same amount of JEN Co., Ltd.), the number of living cells was measured with a hemacytometer (manufactured by Elma Co., Ltd.). As a result, it was confirmed that HepG2 cells formed spheres with the medium composition, and the number of cells also increased to 80,800 cells / mL. Spheres of HepG2 cells formed with the medium composition are shown in FIG.

Reference Test Example 23 Cell Suspension Test Using Cell Line Derived Sphere Ultrapure water (KELKO-CRETE DG, manufactured by Sanyo Co., Ltd.) to a concentration of 0.3% (w / v) ( After being suspended in Milli-Q water), it was dissolved by stirring with heating at 90 ° C. Using this aqueous solution, a DMEM / F-12 medium composition having a final concentration of 0.1% (w / v) was prepared for Daiyutan gum. Also, it is prepared by heating an aqueous solution containing 0.5% (w / v) of native gellan gum (Kelcogel HT, manufactured by San-Ei Gen FFI, Inc.) at 90 ° C., and using this aqueous solution A DMEM / F-12 medium (manufactured by Sigma) composition containing 0.05% and 0.1% (w / v) native gellan gum was prepared.

  Spheres of HeLa cells were prepared using the same method as in Reference Test Example 2, and after adding several tens of spheres to 1 mL of the above-prepared medium, each was allowed to stand at 37 ° C. for 1 hour. The floating state of was visually observed. As a result, it was confirmed that the spheres of HeLa cells were maintained in a suspended state in all of the above medium compositions. Furthermore, it was confirmed that spheres of HeLa cells were sedimented by centrifuging (200 G, 5 minutes) of the cell suspension containing 0.1% (w / v) of diutan gum, and that the cells could be recovered.

Reference Test Example 24: Cell Suspension Test 1 Using Magnetic Beads Having Cell Adhesion Ability
500 μL each of a suspension solution of GEM (registered trademark, Global Eukaryotic Microcarrier, manufactured by GL Sciences Inc.) coated with laminin or fibronectin is dispensed to a 1.5 mL microtest tube (manufactured by Eppendorf) in 500 μL portions, and a magnet stand (TA4899N12) The solvent was removed by accumulating GEM from the GEM suspension solution using Tamagawa Seiki Co., Ltd.). Furthermore, GEM was washed twice with 500 μL of DMEM medium (manufactured by WAKO) containing 10% (v / v) fetal bovine serum, and then suspended in 500 μL of the same medium. This suspension was dispensed at 50 μl per well to Sumilon cell tight plate 24F (manufactured by Sumitomo Bakelite Co., Ltd.) which is a low cell adhesion plate. Subsequently, HepG2 cells prepared separately were added to 250000 cells / mL, and the final volume was adjusted to 500 μL / well in the same medium. After manually stirring the cell suspension, the plate was allowed to stand overnight in a CO ₂ incubator (5% CO ₂ ). After confirming cell adhesion on GEM with a microscope, the cell suspension is transferred to a 1.5 mL microtest tube (manufactured by Eppendorf), and cell adhesion GEM is accumulated using the above-mentioned magnet stand. Qing was removed.

DMEM medium (WAKO) containing 0.015% deacylated gellan gum (KELCOGEL CG-LA, manufactured by Tri Crystal Co., Ltd.) and 10% (v / v) fetal bovine serum using the same method as Reference Test Example 2 Company's composition was prepared. The present medium composition or 1 mL each of the same medium without deacylated gellan gum was added to HepG2 cell-adhered GEM (laminin or fibronectin coating) prepared above, suspended, and then transferred to a Smironceltite plate 24F . Subsequently, the plate is allowed to stand for 6 days in a CO ₂ incubator (5% CO ₂ ), and then the cell culture solution is transferred to a 1.5 mL microtest tube (manufactured by Eppendorf) and placed on the magnet stand. The cell attachment GEM was accumulated while gently pipetting to remove the supernatant. The present GEM was washed once with 1 mL of PBS, 200 μL of trypsin-EDTA (ethylenediaminetetraacetic acid) solution (manufactured by WAKO) was added, and the mixture was incubated at 37 ° C. for 10 minutes. To 200 μL of the cell suspension obtained here, 800 μL of DMEM medium containing 10% (v / v) fetal bovine serum was added, and a portion of the cell suspension was subjected to trypan blue staining (in vitro After adding the same amount of JEN Co., Ltd.), the number of living cells was measured with a hemacytometer (manufactured by Elma Co., Ltd.).

As a result, GEM to which HepG2 cells were attached can be cultured in a suspended state by using the medium composition, and it was confirmed that cells efficiently proliferate in the medium composition. Moreover, it was confirmed that the medium composition was superior in the cell growth promoting effect as compared with the existing medium without deacylated gellan gum. In addition, it was possible to accumulate HepG2 cell-adhered GEM from the medium composition by using a magnetic force, and further confirmed that HepG2 cells could be recovered from the present GEM.
Table 30 shows the cell numbers when HepG2 cells were cultured on GEM for 6 days in a medium containing or not containing deacylated gellan gum. In addition, FIG. 9 shows a suspended state when laminin-coated GEM to which HepG2 cells are attached is cultured with the medium composition.

Reference Test Example 25: Cell floating test 2 using magnetic beads having cell adhesion ability
In the same manner as in Reference Test Example 24, GEM (registered trademark, Global Eukaryotic Microcarrier, manufactured by GL Sciences Inc.) coated with fibronectin was suspended in MF-Medium (registered trademark) mesenchymal stem cell growth medium (manufactured by Toyobo Co., Ltd.). It became cloudy. This suspension was dispensed at 50 μl per well to Sumilon cell tight plate 24F (manufactured by Sumitomo Bakelite Co., Ltd.) which is a low cell adhesion plate. Subsequently, human bone marrow-derived mesenchymal stem cells (manufactured by Cell Applications) prepared separately were added so as to be 250000 cells / mL, and the plate was incubated overnight with CO ₂ incubator (5 The mixture was allowed to stand in% CO ₂ ) to prepare GEM to which mesenchymal stem cells were attached.

MF-Medium (registered trademark) mesenchymal stem cell growth medium (Toyobo) containing 0.015% deacylated gellan gum (KELCOGEL CG-LA, manufactured by Sanyo Co., Ltd.) using the same method as in Reference Test Example 2 The composition of Co., Ltd. was prepared. The present medium composition or 1 mL each of the same medium without deacylated gellan gum was added to the mesenchymal stem cell-adhered GEM (fibronectin coating) prepared above, suspended, and then transferred to a Smilon cell tight plate 24F . Subsequently, the plate is allowed to stand for 4 days in a CO ₂ incubator (5% CO ₂ ), and then the cell culture solution is transferred to a 1.5 mL microtest tube (manufactured by Eppendorf) and placed on the magnet stand. The cell attachment GEM was accumulated while gently pipetting to remove the supernatant. The present GEM was washed once with 1 mL of PBS, 200 μL of trypsin-EDTA (ethylenediaminetetraacetic acid) solution (manufactured by WAKO) was added, and the mixture was incubated at 37 ° C. for 10 minutes. To 200 μL of the cell suspension obtained here, 800 μL of DMEM medium containing 10% (v / v) fetal bovine serum was added, and a portion of the cell suspension was subjected to trypan blue staining (in vitro After adding the same amount of JEN Co., Ltd.), the number of living cells was measured with a hemacytometer (manufactured by Elma Co., Ltd.).

As a result, GEM to which mesenchymal stem cells were adhered can be cultured in a suspended state by using the medium composition, and it was confirmed that cells efficiently proliferate in the medium composition. Moreover, it was confirmed that the medium composition was superior in the cell growth promoting effect as compared with the existing medium without deacylated gellan gum. Moreover, it was possible to accumulate mesenchymal stem cell-adhered GEM from the medium composition by using a magnetic force, and further confirmed that mesenchymal stem cells can be recovered from this GEM.
The number of cells when mesenchymal stem cells are cultured on GEM for 4 days in a medium containing or not containing deacylated gellan gum is shown in Table 31.

Reference Test Example 26 Cell Suspension Test Using Alginate Beads The following test was carried out according to the method of an alginate three-dimensional culture kit manufactured by PG Research, Inc. Add separately prepared HepG2 cells to 2.5 mL of sodium alginate solution (PG Research Co., Ltd.) so as to be 400000 cells / mL, and make human recombinant laminin 511 (Veritas Co., Ltd.) to 5 μg / mL. Then, a cell suspension was prepared. The cell suspension was collected by a 5 mL syringe (manufactured by Terumo Corporation) equipped with a sonde, and then a 22 G injection needle (manufactured by Terumo Corporation) was mounted on the syringe. Subsequently, 10 drops of the cell suspension were added to the wells of a 24-well flat-bottomed microplate (PG Research, Inc.) to which 2 mL of an aqueous calcium chloride solution (PG Research, Inc.) was added. After standing at room temperature for 10 minutes to confirm formation of alginic acid beads, the calcium chloride solution was removed, 2 mL of PBS was added, and the mixture was allowed to stand at room temperature for 15 minutes. Furthermore, after removing PBS, 2 mL of DMEM medium (manufactured by WAKO) containing 10% (v / v) fetal bovine serum was added and allowed to stand at room temperature for 15 minutes. After removing the medium, 0.03% deacylated gellan gum (KELCOGEL CG-LA, manufactured by Tri Crystal Co., Ltd.) and 10% (v / v) fetal bovine were prepared using the same method as in Reference Test Example 2. Add 1 mL each of the medium composition of DMEM medium containing serum (manufactured by WAKO) or the same medium without deacylated gellan gum to each well, and allow to stand in a CO ₂ incubator (5% CO ₂ ) for 8 days Cultured. Medium change was performed on the fourth day of culture.

  After transferring the cultured alginate beads to a 1.5 mL microtest tube (manufactured by Eppendorf) using a 1 mL chip, add 1 mL of sodium citrate solution (PG Research Co., Ltd.) to each tube, and room temperature The alginate beads were dissolved by stirring for 15 minutes. Subsequently, cells were sedimented by centrifugation at 300 G for 3 minutes, and the supernatant was removed. To the cells, 200 μL of a trypsin-EDTA (ethylenediaminetetraacetic acid) solution (manufactured by WAKO) was added, and incubated at 37 ° C. for 5 minutes. To 200 μL of the cell suspension obtained here, 800 μL of DMEM medium containing 10% (v / v) fetal bovine serum was added, and a portion of the cell suspension was subjected to trypan blue staining (in vitro After adding the same amount of JEN Co., Ltd.), the number of living cells was measured with a hemacytometer (manufactured by Elma Co., Ltd.).

As a result, it was possible to culture the alginate beads in which HepG2 cells were embedded in a suspended state by using the medium composition, and it was confirmed that the cells proliferated efficiently with the medium composition. Moreover, it was confirmed that the medium composition was superior in the cell growth promoting effect as compared with the existing medium without deacylated gellan gum.
Table 32 shows the cell numbers when HepG2 cells were cultured in alginate beads for 8 days in a medium containing or not containing deacylated gellan gum. In addition, Fig. 10 shows the suspended state when alginate beads in which HepG2 cells are embedded are cultured with the medium composition.

Reference test example 27: Cell suspension test using collagen gel capsule A: Collagen Cellmatrix (registered trademark) Type IA (cell matrix, Nitta Gelatin Co., Ltd.) for tissue culture, B: DMEM / F- at 10-fold concentration 12 medium (manufactured by Aldrich), C: reconstitution buffer (2.2 g of sodium hydrogen carbonate in 100 mL of 0.05 N sodium hydroxide solution, HEPES (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid)); Each of 77 g added and filter-sterilized) was mixed so that it became A: B: C = 8: 1: 1, cooling in ice. Furthermore, human recombinant laminin 511 (manufactured by Veritas, Inc.) was added to a concentration of 5 μg / mL to prepare 500 μL of a collagen mixed solution. Add separately prepared HepG2 cells to the mixed solution to 200,000 cells / mL, and use the 1.5 mL syringe (manufactured by Terumo Corporation) fitted with a 25G injection needle (manufactured by Terumo Corporation). It was collected. Subsequently, using the above-described syringe, a flat-bottomed tube (manufactured by BM Instruments Co., Ltd.) to which 10 mL of DMEM medium (manufactured by WAKO) containing 10% (v / v) fetal bovine serum previously incubated at 37 ° C. The cell suspension was dropped one by one. After incubating in a 37 ° C. water bath for 10 minutes to confirm the formation of an irregularly shaped collagen gel capsule having a diameter of about 2 mm, deconjugation to a final concentration of 0.04% by the same method as Reference Test Example An acylated gellan gum (KELCOGEL CG-LA, manufactured by Three Crystal Co., Ltd.) was added, and the mixture was gently stirred to suspend the above capsule. Subsequently, the tube was statically cultured in a CO ₂ incubator (5% CO ₂ ) for 5 days.

  25 mL of PBS was added to the culture solution containing the collagen gel capsule, the collagen gel capsule was sedimented by centrifugation at 400 G for 5 minutes, and the supernatant was removed. Again, 25 mL of PBS was added and centrifugation was performed, and the supernatant was removed so that the remaining amount became 5 mL. After adding 20 μl of 1% (W / V) collagenase L (manufactured by Nitta Gelatin Co., Ltd.) to the solution, the mixture was shaken at 37 ° C. for 2 hours. After confirming the dissolution of the collagen gel, 10 mL of PBS was added, cells were sedimented by centrifugation at 400 G for 5 minutes, and the supernatant was removed. 1 mL of a trypsin-EDTA (ethylenediaminetetraacetic acid) solution (manufactured by WAKO) was added to the cells, and incubated at 37 ° C. for 5 minutes. To the cell suspension thus obtained, 4 mL of DMEM medium containing 10% (v / v) fetal bovine serum was added, cells were sedimented by centrifugation at 400 G for 5 minutes, and the supernatant was removed. The obtained cells are suspended in 2 mL of the above medium, and an equal amount of trypan blue staining solution (manufactured by Invitrogen) is added to a portion of the suspension, and then the cells are counted with a hemocytometer (manufactured by Elma Sales Co., Ltd.) The number of viable cells was measured.

As a result, it was possible to culture collagen gel capsules in which HepG2 cells were embedded in a suspended state by using the medium composition, and it was confirmed that the cells proliferated efficiently in the medium composition. Moreover, it was confirmed that the medium composition was superior in the cell growth promoting effect as compared with the existing medium without deacylated gellan gum.
The number of cells when HepG2 cells were cultured in a collagen gel capsule for 5 days in a medium containing or not containing deacylated gellan gum is shown in Table 33. In addition, a suspended state when a collagen gel capsule in which HepG2 cells are embedded is cultured with the medium composition is shown in FIG.

Reference Test Example 28: Recovery test of spheres using filter 0.015% deacylated gellan gum (KELCOGEL CG-LA, manufactured by Three Crystal Co., Ltd.) and 10% (v) using the same method as in Reference Test Example 2. / V) A composition of DMEM medium (manufactured by WAKO) containing fetal bovine serum was prepared. As a control, the same medium without deacylated gellan gum was prepared. Spheres of HepG2 cells were prepared using the same method as in Reference Test Example 2, and after adding the spheres to 1 mL of the culture medium prepared above so that the number of each of 86000 cells was obtained, the cells were allowed to stand at 37 ° C for 1 hour Then, the floating state of sphere cells was visually observed. Furthermore, the cell suspension was added on a cell strainer (manufactured by Becton Dickinson) having a mesh size of 40 μm, and the spheres were trapped on the filter. Subsequently, the spheres were collected into a 15 mL tube by pouring 10 mL of PBS from the back of the filter, and the spheres were sedimented by centrifugation at 300 G for 5 minutes. After removing the supernatant, 500 μL of a trypsin-EDTA (ethylenediaminetetraacetic acid) solution (manufactured by WAKO) was added to the spheres and incubated at 37 ° C. for 5 minutes. 1 mL of DMEM medium containing 10% (v / v) fetal bovine serum was added to the cell suspension obtained here, and trypan blue staining solution (manufactured by Invitrogen Co., Ltd.) was partially added to the cell suspension. After adding the amount, the number of living cells was measured with a hemocytometer (manufactured by Elma Sales Co., Ltd.). As a result, it was confirmed that the spheres of HepG2 cells were maintained in suspension in the above medium composition. Furthermore, by filtering this sphere suspension containing 0.015% deacylated gellan gum, it is possible to recover the sphere of HepG2 cells at a recovery rate equivalent to the medium without deacylated gellan gum. It was confirmed. Table 34 shows the relative number of cells recovered from the medium containing the deacylated gellan gum, assuming that the number of HepG2 cells collected by the filter is 1 using a medium not containing the deacylated gellan gum.

Reference Test Example 29 Cell Suspension Test of Spheres Using Admixtures of Various Polysaccharides Using the same method as Reference Test Example 9, xanthan gum (KELTROL CG, manufactured by Tri Crystal Co., Ltd.), sodium alginate (Duck alginate NSPM, Food Chemifa), Locust bean gum (GENUGUM RL-200-J, Sanki Co., Ltd.), methylcellulose (cP400, WAKO Co.), κ-carrageenan (GENUGEL WR-80-J, Sanyo Co., Ltd.) ), Pectin (GENU pectin LM-102AS, manufactured by Tricrystal Co., Ltd.) or diutatan gum (KELCO CRETE DG-F, manufactured by Tricrystal Co., Ltd.) and deacylated gellan gum (KELCOGEL CG-LA, manufactured by Tricrystal Co., Ltd.) DMEM / F-12 medium set combined and mixed The product was prepared. Spheres of HepG2 cells were prepared using the same method as in Reference Test Example 2, and after adding several tens of spheres to 1 mL of the culture medium prepared above, each was allowed to stand at 37 ° C. for 1 hour and 1 The floating state of sphere cells after night was visually observed. As a result, it was confirmed that the spheres of HepG2 cells were maintained in a suspended state in all of the above medium compositions. Furthermore, after adding 2 volumes of medium, the cell suspension was centrifuged (500 G, 5 minutes) to precipitate the spheres of HepG2 cells, and it was confirmed in all medium compositions that cells could be recovered. . When the dispersion state of the sphere after one night was visually confirmed, the results obtained by evaluating the floating dispersion state as 沈降, partial sedimentation / dispersion state as Δ, and the sedimentation state as x are shown in Tables 35 and 36. In addition,-in a table | surface shows unimplemented.

Bead and Cell Dispersion Comparison 1
Regarding the deacylated gellan gum-containing medium and the methylcellulose-containing medium prepared in the above (comparative example), the dispersion state of dextran beads Cytodex (registered trademark) 1 (manufactured by GE Healthcare Life Sciences) and HeLa cell spheres were compared. The results are shown in the tables (Tables 37 and 38). Because the dispersion state of Cytodex1 and HeLa cell spheres is well correlated, Cytodex1 can be used as a cell sphere model.

Dispersion of beads and cells 2
With respect to the polysaccharide- and deacylated gellan gum-containing medium prepared in Reference Test Example 9, dispersion states of polystyrene beads (Size 500-600 μm, manufactured by Polysciences Inc.) and HepG2 cell spheres were compared. The floating dispersion state was evaluated as ○, the partial sedimentation / dispersion state was Δ, and the sedimentation state was evaluated as x. The results are shown in the table (Table 39). Since the dispersion state of polystyrene beads and HepG2 cell spheres is well correlated, polystyrene beads can be used as a cell sphere model.

Reference test example 30: Suspension culture test of plant callus derived from rice 50 ripened seeds (purchased from Koto Agricultural Cooperative Association) of rice Nipponbare carefully selected by salt water selection are transferred to a 50 mL polystyrene tube (manufactured by BD Falcon) and sterilized water After washing with 50 mL, it was stirred for 1 minute in 30 mL of 70% ethanol water. After the ethanol water was removed, 30 mL of Kitchen Heighter (manufactured by Kao Corporation) was added and stirred for 1 hour. After removing the kitchen heater, it was washed 4 times with 50 mL of sterile water. Seeds sterilized here are 1.5 mL / well (24 mL of Murashige Skoog basal medium (M 9274, Sigma Aldrich), containing 2 μg / mL of 2,4-dichlorophenoxyacetic acid (Sigma Aldrich) and agar). The plate was placed on a well flat bottom microplate (manufactured by Corning). The cells were cultured at 30 ° C. for 16 hours in the dark / 8 hours in the dark for 3 weeks, and cream colored calli (1 to 2 mm) grown on the scutella of the seeds were collected.

  After suspending the deacylated gellan gum (KELCOGEL CG-LA, manufactured by Sanyo Co., Ltd.) to 0.3% (w / v) in ultrapure water (Milli-Q water), heating at 90 ° C. The solution was dissolved by stirring, and the aqueous solution was autoclaved at 121 ° C. for 20 minutes. Using this solution, the final concentration is 0.03% (w / m) in Murashige Skoog basal medium (M 9274, Sigma Aldrich) containing 2 μg / mL 2,4-dichlorophenoxyacetic acid (Sigma Aldrich). A medium composition was prepared to which v) deacylated gellan gum was added. The 15 callus prepared above were added to 10 mL of the medium composition / flat-bottomed tube (manufactured by BM Instruments), and shake culture was performed at 25 ° C. for 7 days. As a result, it has been confirmed that rice-derived calli can be cultured in a floating state by using the medium composition, and the callus is maintained by the medium composition. The floating state when rice-derived calli are cultured with the medium composition is shown in FIG.

[Production Example 1: Production of crystalline cellulose-derived cellulose nanofibers]
After 4 parts by mass of commercially available crystalline cellulose (PH-101 manufactured by Asahi Kasei Chemicals Co., Ltd.) is dispersed by adding 396 parts by mass of pure water, the pressure is reduced to 220 MPa using a high pressure pulverizer (starburst system) manufactured by Sugino Machine Co., Ltd. The mixture was ground 100 times to obtain an aqueous dispersion (MNC) of crystalline nanofibers derived from cellulose nanofibers. The obtained dispersion was measured in a petri dish, dried at 110 ° C. for 1 hour, water was removed, the amount of residue was measured, and the concentration was measured. As a result, the cellulose concentration (solid content concentration) in water was 1.0% by mass. The aqueous dispersion was autoclaved at 121 ° C. for 20 minutes for sterilization.

[Production example 2: Production of pulp-derived cellulose nanofibers]
After adding 145 parts by mass of pure water to 5 parts by mass of a commercially available kraft pulp (LBKP made by Oji F-Tex Co., Ltd., solid content 89% by mass) and dispersing it, a stone mill type pulverizer (mass colloider) manufactured by Masuko Sangyo Co., Ltd. Pulverization processing was performed 9 times at 1500 rpm using it, and pulp slurry was produced. An aqueous dispersion (PNC) of nanocellulose was obtained by treating the pulp slurry 300 times at 220 MPa using a high-pressure pulverizer (star burst system) manufactured by Sugino Machine Co., Ltd. The obtained dispersion was measured in a petri dish, dried at 110 ° C. for 1 hour, water was removed, the amount of residue was measured, and the concentration was measured. As a result, the cellulose concentration (solid content concentration) in water was 1.6% by mass. The aqueous dispersion was autoclaved at 121 ° C. for 20 minutes for sterilization.

[Production example 3: Production of chitin nanofibers]
20 parts by mass of commercially available chitin powder (manufactured by Koyo Chemical Co., Ltd.) is added and dispersed in 980 parts by mass of pure water, and then dispersed using Sugino Machine Co., Ltd. high pressure pulverizer (star burst system) to 200 at 245 MPa. Pulverization processing was performed to obtain an aqueous dispersion (CT) of chitin nanofibers. The obtained dispersion was measured in a petri dish, dried at 110 ° C. for 1 hour, water was removed, the amount of residue was measured, and the concentration was measured. As a result, the chitin concentration (solid content concentration) in water was 2.0% by mass. The aqueous dispersion was autoclaved at 121 ° C. for 20 minutes for sterilization.

[Test Example 1: Measurement of Average Fiber Diameter D and Average Fiber Length L]
The average fiber diameter (D) of the nanofibers was determined as follows. First, the nanofiber dispersion prepared in Production Examples 1 to 3 was subjected to a hydrophilization treatment for 3 minutes with the ion cleaner (JIC-410) manufactured by Nippon Denshi Co., Ltd. for the collodion support film manufactured by SOK Inc. Solution was added dropwise and dried at room temperature. This is observed with a transmission electron microscope (TEM, H-8000) (10,000 times) manufactured by Hitachi, Ltd. at an acceleration voltage of 200 kV, and using the obtained image, the number of samples: 200 to 250 The fiber diameter of each of the nanofibers was measured, and the number average value was taken as the average fiber diameter (D).
In addition, the average fiber length (L) was such that the nanofiber dispersion prepared in the production example was diluted with pure water to 100 ppm, and the nanofibers were uniformly dispersed using an ultrasonic cleaner. This nanofiber dispersion was cast onto a silicon wafer whose surface had been hydrophilized in advance using concentrated sulfuric acid, and dried at 110 ° C. for 1 hour to prepare a sample. Using an image of the obtained sample observed with a scanning electron microscope (SEM, JSM-7400F) (2,000 ×) manufactured by Nippon Denshi Co., Ltd., the number of samples: 150 to 250, one for each nanofiber. The fiber length of the book was measured, and the number average value was taken as the average fiber length (L).
The average fiber diameter D and the average fiber length L of the nanofibers obtained in Production Examples 1 to 3 were determined, and the aspect ratio L / D was determined from these values. The obtained results are shown in Table 40.

[Examples 1 to 4]
Using the nanofiber dispersion solution and the deacylated gellan gum aqueous solution prepared in the above-mentioned Production Example 1 to Production Example 3, media compositions shown in Table 41 below were prepared.
First, sterile water was added to the cellulose nanofibers MNC, PNC and chitin nanofibers prepared in Production Examples 1 to 3 to dilute them to 1% (w / v) aqueous dispersion. On the other hand, 99 parts by volume of sterile water is added to 1 part by mass of deacylated gellan gum (KELCOGEL CG-LA, manufactured by Tri Crystal Co., Ltd .: DAG) and dissolved and sterilized by autoclaving at 121 ° C. for 20 minutes. An aqueous solution of acylated gellan gum 1% (w / v) was prepared.
One volume part of the 1% (w / v) dispersion or aqueous solution described above was placed in a 50 mL conical tube, and 49 volume parts sterile water was added and pipetted until uniform. To this, 50 parts by volume of double concentrated DMEM (high glucose, manufactured by Aldrich, containing a predetermined amount of sodium hydrogen carbonate) sterile filtered with a 0.22 μm filter is added, mixed by pipetting, and the nanofiber concentration is 0 .01% (w / v) of the medium composition was adjusted.
Similarly, a medium composition was prepared by adding a nanofiber dispersion or a deacylated gellan gum aqueous solution to a desired concentration of 0.01 to 0.1% (w / v) in the final concentration.

[Example 5 and Comparative Examples 2 to 5]
κ-carrageenan (GENUGEL WR-80-J, manufactured by Three Crystal Co., Ltd .: Car) (Example 5), locust bean gum (GENUGUM RL-200-J, manufactured by Three Crystal Co., Ltd .: LBG) (Comparative Example 2), Xanthan gum (KELTROL CG, manufactured by Tri Crystal Co., Ltd .: Xan) (Comparative Example 3), Daitan gum (KELCO CRETE DG-F, manufactured by Tri Crystal Co., Ltd .: DU) (Comparative Example 4), Na alginate (Duck alginate NSPM, 99 parts by weight of sterile water was added to 1 part by weight of Food Chemifa Co., Ltd. (Comparative Example 5) (Comparative Example 5), and the solution was sterilized by autoclaving at 121 ° C. for 20 minutes.
With respect to the polysaccharide solution prepared as described above, the polysaccharide solution is added to a final concentration of 0.03, 0.05, 0.07, 0.1% (w / v) by the same operation as in Examples 1 to 4. The prepared medium composition was prepared.

[Test Example 2: Evaluation of floating action 1]
Polystyrene beads (manufactured by Polysciences Inc., 200 to 300 μm) were added to the medium compositions of Examples 1 to 5 and Comparative Examples 2 to 5, and the beads were uniformly dispersed in the medium composition by vortexing. After confirmation, it was allowed to stand at room temperature (25 ° C.) for one day, and the dispersion state of the beads was visually confirmed. The state in which beads were uniformly suspended in the medium composition was evaluated as ◎, the state in which a part of the supernatant was generated as ○, and the state of sedimentation as x. The results are shown in Table 41.

  As a result, the medium compositions of Examples 1 to 4 showed the action of suspending the beads. Further, in Example 5, although the floating effect of the beads was observed at room temperature, the beads were sedimented by heating to 37 ° C., and the floating effect was not obtained under the cell culture conditions. In Comparative Examples 2 to 5, the beads were completely sedimented to the bottom.

  * The −-carrageenan (Car) of Example 5 showed a floating action at 25 ° C, but immediately lost the floating action and settled at 37 ° C equivalent to the cell culture conditions. For the other media, identical results were obtained at 37 ° C and 25 ° C.

[Test Example 3: Evaluation 2 of floating action]
The floating action in the low concentration range (0.01 to 0.04% (w / v)) of the culture medium compositions of Examples 2, 4 and 5 and Comparative Example 2 was evaluated in detail in the same manner as in Test Example 2. . Polystyrene beads were added, and after standing for 2 days, the dispersed state of the beads was visually confirmed. The floating dispersion state was evaluated as ○, and the sedimentation state was evaluated as x. For partial sedimentation / dispersion, the bead floating rate was calculated based on the height of the floating area in a 10 mL conical tube. The results are shown in Table 42.

  ※ indicates bead floating rate. PNC showed floating action at a concentration of 0.015% (w / v) or more, and the medium composition of Example 4 at a concentration of 0.015% (w / v) or more. In the medium composition of Example 2, the floating action gradually improved as the concentration increased. The medium composition of Example 5 exhibited a floating action at 25 ° C. at 0.02% or more, but immediately lost the floating action at 37 ° C. and settled (*). For the other media, identical results were obtained at 37 ° C and 25 ° C.

[Test Example 4: Viscosity of Medium Composition]
The viscosities of the medium compositions of Examples 1 to 5 and Comparative Examples 2 to 5 were evaluated using tuning fork vibrational viscosity measurement (SV-1A, A & D Company Ltd.) under 25 ° C. conditions. The results are shown in FIG. As a result, since the medium composition of the present invention has an extremely low content of nanofibers or polysaccharide thickener, no significant increase in viscosity is observed as compared to the viscosity of a general medium. The From the comparison with the results of Test Example 2, no correlation was found between the viscosity and the floating action.

[Test Example 5: Scanning Electron Microscopy of Media Composition]
The medium compositions prepared in Examples 1 to 5 and Comparative Examples 3 to 4 are cast onto silicon wafers whose surfaces have been hydrophilized in advance using concentrated sulfuric acid, and the solution is heated at 110 ° C. (only Comparative Example 1 at room temperature) for 1 hour After drying, it was rinsed with pure water to remove excess salts and the like, and then dried again at 110 ° C. for 1 hour to obtain a sample. The aforementioned sample was observed using a scanning electron microscope (SEM, JSM-7400F, 10,000 times) manufactured by JEOL. The observation results of the culture medium compositions of Examples 1 to 4 and Comparative Examples 3 to 4 are shown in FIGS. 14 to 21.

  As a result of observation, in Examples 1 to 4 and Example 5 dried at room temperature, a large number of fibers were observed, whereas in Examples 5 and Comparative Examples 3 to 4 dried at 110 ° C. It was not observed. The spherical objects observed in large numbers in the observation image are those in which the salt component in the culture medium is deposited. This result suggested that the fiber structure contained in the medium composition may contribute to the floating action.

[Test Example 7: Floating Effect of Sphere]
The human hepatoma cell line HepG2 (DS Pharma Biomedical Co., Ltd.) is suspended in DMEM medium (WAKO) containing 10% (v / v) fetal bovine serum to 50,000 cells / mL, and the suspension is After 10 mL was seeded on EZ SPHERE (manufactured by Asahi Glass Co., Ltd.), the cells were cultured for 2 days in a CO ₂ incubator (5% CO ₂ ). 80 mL of the suspension of spheres obtained here was centrifuged (800 rpm, 5 minutes) to sediment the spheres, and the supernatant was removed to prepare 4.5 mL of sphere suspension. Subsequently, 10 mL each of the medium compositions of Examples 1 to 4 and Comparative Example 1 and Comparative Examples 3 to 5 were placed in 15 mL conical tubes, and 100 μL of sphere suspension of HepG2 cells was further added. The spheres were dispersed by pipetting, incubated at 37 ° C. for 5 days, and the dispersion state of the spheres in the medium composition was visually observed. The state in which the spheres were uniformly suspended in the medium composition was evaluated as ◎, the state in which the supernatant was generated as ○, and the state of sedimentation as x. The observation results of the medium compositions of Examples 1 to 5 and Comparative Examples 3 to 5 are shown in Table 43 and FIGS. 22 to 29.

  As a result, in Examples 1 to 4, the medium composition was in a floating state even after 6 days of culture. On the other hand, in the medium compositions of Example 5 and Comparative Examples 3 to 5, all the spheres were sedimented, and the spheres were further aggregated.

[Example 1 'to Example 4']
A 1% (w / v) aqueous dispersion was prepared by adding sterile water to the cellulose nanofibers MNC, PNC and chitin nanofibers prepared in Production Examples 1 to 3, respectively. On the other hand, 99 parts by volume of sterile water is added to 1 part by mass of deacylated gellan gum (KELCOGEL CG-LA, manufactured by Tri Crystal Co., Ltd .: DAG) and dissolved and sterilized by autoclaving at 121 ° C. for 20 minutes; % (W / v) aqueous solution was prepared. Using the 1% (w / v) fiber dispersion prepared above or the deacylated gellan gum aqueous solution, the final concentration is 0.01%, 0.03%, 0.06% and 0.1% (w / w). The medium composition was prepared by adding it to DMEM medium (high-glucose, manufactured by Nissui Pharmaceutical Co., Ltd.) containing 10% (v / v) fetal bovine serum to be v).

[Example 5 'and Comparative Examples 3' to 5 ']
κ-carrageenan (GENUGEL WR-80-J, manufactured by Three Crystal Co., Ltd .: Car), xanthan gum (KELTROL CG, manufactured by Three Crystal Co., Ltd .: Xan), Daiutan gum (KELCO CRETE DG-F, manufactured by Three Crystal Co., Ltd.): 99% by volume of sterilized water is added to 1 part by weight of sodium alginate (Duck alginic acid NSPM, manufactured by Food Chemifa: Alg) and dissolved and sterilized by autoclaving at 121 ° C. for 20 minutes, each 1% (w / V) An aqueous polysaccharide solution was prepared. About each polysaccharide aqueous solution prepared above, final concentration 0.01% and 0% to DMEM medium (manufactured by Nissui Pharmaceutical Co., Ltd.) containing 10% (v / v) fetal bovine serum by the same operation as in Examples 5 to 8. An aqueous polysaccharide solution was added to .03%, 0.06% and 0.1% (w / v) to prepare a medium composition.

[Test Example 8: Cell Proliferation Test]
The human breast cancer cell line MCF-7 (manufactured by DS Pharma Biomedical Co., Ltd.) and the human melanoma cell line A375 (manufactured by ATCC) were subjected to Examples 1 'to 5' and Comparative Examples 3 'to 33333 cells / mL. After seeding with the medium composition prepared in Comparative Example 5 ', 150 μL per well was distributed to the wells of a 96-well flat bottom ultra-low adhesion surface microplate (Corning # 3474). As negative control, MCF7 cells or A375 cells suspended in the same medium without nanofibers or polysaccharides were dispensed. Subsequently, the plate was incubated in a CO ₂ incubator (37 ° C., 5% CO ₂ ) for up to 6 days. For two days and six days culture solution after culture, after ATP reagent ^{150μL (CellTiter-Glo TM Luminescent Cell} Viability Assay, Promega Corp.) was added and suspended and allowed to stand at room temperature for about 10 minutes, FlexStation3 ( The luminescence intensity (RLU value) was measured by Molecular Devices, Inc., and the number of living cells was determined by subtracting the luminescence value of the medium alone.

  As a result, the medium composition containing PNC, MNC, and nanochitin can be cultured in a uniformly dispersed state without becoming excessively large in cell aggregate size, and proliferate efficiently. Was confirmed. On the other hand, no growth promotion was observed in the medium composition containing sodium alginate. Tables 44 to 47 show the RLU values (ATP measurement, luminescence intensity) after 2 days and 6 days of static culture of MCF7 cells, and the results of A375 cells are shown in FIGS. 30 to 33 after 6 days. The RLU values after 48 to 51 and after 6 days are shown in FIG. 34 to FIG. The results of MCF7 cells are shown in FIG. 38 and the results of A375 cells are shown in FIG.

[Test Example 9: Storage test using 3T3-L1 cells]
The mouse preadipocyte cell line 3T3-L1 (manufactured by ATCC) was seeded on a 10 cm polystyrene Petri dish using DMEM medium containing 10% FBS, and cultured in an incubator set at 37 ° C., 5% CO ₂ . After the 3T3-L1 cells become confluent, the medium is aspirated and removed, and the FBS is removed with D-PBS (manufactured by Wako Pure Chemical Industries, Ltd.), 1 ml of a solution containing 0.25% Trypsin and 1 mM EDTA (manufactured by Wako Pure Chemical Industries, Ltd.) Was added to the above polystyrene petri dish. After confirmation of cell detachment, 10% by volume of FBS-containing DMEM medium was added to recover the cells from the petri dish and transferred to a centrifuge tube. After centrifugation at 300 × g, the supernatant was removed. 100 μl of cell suspension was added to a 1.5 mL microtube as a cell suspension of approximately 100 × 10 ⁴ cells / mL, and it had been previously prepared to contain 10% (v / v) FBS 100 μL each of the medium compositions of Examples 1 to 2 and Examples 4 to 5 and Comparative Examples 3 and 5 were added, and a cell suspension was prepared by pipetting.
The cells are stored in a sealed state for 10 days at room temperature under room temperature, and a portion of the cell suspension after 3 days and 10 days is diluted 1/10 with 10% FBS-containing DMEM medium to dilute the cell suspension liquid 100 [mu] L of ATP reagent ^{100μL (CellTiter-Glo TM luminescent Cell} Viability Assay, Promega Corp.) was added to suspend the, was allowed to stand at room temperature for 15 minutes, the emission intensity (RLU value at FlexStation3 (Molecular Devices Corp.) ) Was measured, and the number of viable cells was determined by subtracting the luminescence value of the medium alone. Negative controls were samples of medium only without polysaccharide.

  As a result, while the cell viability of each of the medium compositions of the negative control or Comparative Example 3 and Comparative Example 5 was significantly reduced in ATP value at room temperature storage for 3 to 10 days, Example 1 to Example 2 And in the medium composition of Example 4, the fall of the ATP level was suppressed and the cytoprotective effect was shown. The results of the number of viable cells are shown in Table 52.

[Test Example 10: Storage test using CHO-K1 cells]
Chinese hamster ovary strain CHO-K1-hIFNβ cells (transferred from Prof. Kawahara, Kitakyushu National College of Technology) were seeded on 10 cm polystyrene dishes using F12 medium containing 10% FBS, and set to 5% CO ₂ at 37 ° C. The cells were cultured in an incubator. While the CHO-K1-hIFNβ cells become confluent, the medium is aspirated and removed, and the FBS is removed with D-PBS (manufactured by Wako Pure Chemical Industries, Ltd.), 1 ml of a solution containing 0.25% Trypsin and 1 mM EDTA (Wako Pure Chemical Industries, Ltd. Made in the above polystyrene petri dish. After confirmation of cell detachment, 10% FBS-containing F12 medium was added to recover the cells from the petri dish and transferred to a centrifuge tube. After centrifugation at 300 × g, the supernatant was removed. Example 2 in which 25 μL of cell suspension was added to a 1.5 mL microtube as a cell suspension of approximately 5 × 10 ⁶ cells / mL, and previously prepared to contain 10% (v / v) FBS A cell suspension was prepared by adding 25 μL each of the medium composition of Example 4 and pipetting. The portion of the cell suspension after storage for one day at room temperature in a sealed state was 1/10 diluted with 10% FBS-containing F12 medium, ATP reagent 100μL cell suspension 100μL of the diluted ^{(CellTiter-Glo} TM Luminescent Add Cell Viability Assay (manufactured by Promega), suspend it, and leave it at room temperature for about 10 minutes. Measure the luminescence intensity (RLU value) with FlexStation 3 (Molecular Devices), and measure the luminescence value of the medium alone. The number of viable cells was determined by subtraction. Negative controls were samples of medium only without polysaccharide.

  As a result, each cell viability in the negative control decreased ATP value when stored at room temperature for 1 day, whereas the medium compositions of Example 2 and Example 4 showed ATP level at seeding level, It showed a protective effect. The results of the number of viable cells are shown in Table 53.

[Test Example 11: Storage test using 3T3-L1 cells, concentration change of polysaccharide]
The mouse preadipocyte cell line 3T3-L1 (manufactured by ATCC) was seeded on a 10 cm polystyrene Petri dish using DMEM medium containing 10% FBS, and cultured in an incubator set at 37 ° C., 5% CO ₂ . After the 3T3-L1 cells become 40% confluent, the medium is aspirated and removed, and the FBS is removed with D-PBS (manufactured by Wako Pure Chemical Industries, Ltd.), 1 ml of a solution containing 0.25% Trypsin and 1 mM EDTA (Wako Pure Chemical Industries, Ltd. Made in the above polystyrene petri dish. After confirmation of cell detachment, 10% by volume of FBS-containing DMEM medium was added to recover the cells from the petri dish and transferred to a centrifuge tube. After centrifugation at 300 × g, the supernatant was removed. 100 μl of cell suspension was added to a 1.5 mL microtube as a cell suspension of approximately 100 × 10 ⁴ cells / mL, and it had been previously prepared to contain 10% (v / v) FBS Cell suspensions were prepared by adding 100 μL each of the medium compositions different in the concentration of the polysaccharides of Example 2 and Example 4 and Comparative Example 5 and pipetting them.
The cells were stored still for 8 days at room temperature in a sealed state, and a portion of the cell suspension after 0, 5, and 8 days was diluted 1/5 in DMEM medium containing 10% FBS and diluted. cell suspension 100 [mu] L of ATP reagent ^{100μL (CellTiter-Glo TM luminescent cell} Viability Assay, Promega Corp.) was added to suspend the, was allowed to stand at room temperature for 15 minutes, the emission intensity at FlexStation3 (Molecular Devices Corp.) (RLU value) was measured, and the number of viable cells was determined by subtracting the luminescence value of the medium alone. Negative controls were samples of medium only without polysaccharide.

  As a result, while the cell viability of each of the medium compositions of the negative control or Comparative Example 5 was significantly reduced in ATP value after storage for 5 to 8 days at room temperature, the medium compositions of Example 2 and Example 4 were Showed a cytoprotective effect, because the decrease in ATP level was suppressed. The results of the number of viable cells are shown in Table 54.

[Test Example 12: Effect on cell survival action of MDCK cells]
After suspending the deacylated gellan gum (KELCOGEL CG-LA, manufactured by Sanyo Co., Ltd.) to 0.3% (w / v) in ultrapure water (Milli-Q water), heating at 90 ° C. The solution was dissolved by stirring, and the aqueous solution was autoclaved at 121 ° C. for 20 minutes. The final concentration of 0.005% (w / v) and 0.015% deacylated gellan gum was added to EMEM medium (Wako Pure Chemical Industries, Ltd.) containing 10% (v / v) fetal bovine serum using this solution Media compositions or non-supplemented media compositions without deacylated gellan gum were prepared. Subsequently, the canine renal tubular epithelial cell line MDCK (DS Pharma Biomedical Co., Ltd.) cultured overnight on a medium from which serum has been removed (stavation treatment) is deacylated gellan gum described above so as to have 100,000 cells / mL. After seeding with the added medium composition, 100 μL per well was distributed to the wells of a 96-well flat bottom ultra-low adhesion surface microplate (Corning # 3474). Each plate was cultured at rest in a CO ₂ incubator (37 ° C., 5% CO ₂ ) and continued for 15 days. 2, 6, 9, 12, 15 days of ATP reagent ^{100μL (CellTiter-Glo TM Luminescent Cell} Viability Assay, Promega Corp.) with respect to culture solution was added to suspend the, was allowed to stand at room temperature for about 10 minutes The luminescence intensity (RLU value) was measured with FlexStation 3 (manufactured by Molecular Devices), and the number of living cells was determined by subtracting the luminescence value of the medium alone.

  As a result, when MDCK cells were cultured on a low adhesion plate using the medium composition of the present invention, it became clear that the decrease in the number of viable cells was suppressed. The RLU values (ATP measurement, luminescence intensity) in each culture are shown in Table 55.

[Test Example 13: Effect on cell survival action of Vero cells]
After suspending the deacylated gellan gum (KELCOGEL CG-LA, manufactured by Sanyo Co., Ltd.) to 0.3% (w / v) in ultrapure water (Milli-Q water), heating at 90 ° C. The solution was dissolved by stirring, and the aqueous solution was autoclaved at 121 ° C. for 20 minutes. A medium composition prepared by adding a final concentration of 0.005% (w / v) and 0.015% deacylated gellan gum to Emedium 199 medium (manufactured by Sigma) containing 5% (v / v) fetal bovine serum using this solution A non-supplemented medium composition was prepared which did not contain any substance or deacylated gellan gum. Subsequently, the above-mentioned deacylated gellan gum was added to a monkey kidney epithelial cell line Vero (made by DS Pharma Biomedical Co., Ltd.) cultured overnight for 1 day (stavation treatment) in a medium from which serum was removed (100 cells / mL). After seeding with the medium composition, 100 μL per well was distributed to the wells of a 96-well flat bottom ultra low adhesion surface microplate (Corning # 3474). Each plate was cultured at rest in a CO ₂ incubator (37 ° C., 5% CO ₂ ) and continued for 15 days. 2, 6, 9, 12, 15 days of ATP reagent ^{100μL (CellTiter-Glo TM Luminescent Cell} Viability Assay, Promega Corp.) with respect to culture solution was added to suspend the, was allowed to stand at room temperature for about 10 minutes The luminescence intensity (RLU value) was measured with FlexStation 3 (manufactured by Molecular Devices), and the number of living cells was determined by subtracting the luminescence value of the medium alone.

  As a result, when Vero cells were cultured on a low adhesion plate using the medium composition of the present invention, it became clear that the decrease in the number of viable cells was suppressed. The RLU values (ATP measurement, luminescence intensity) in each culture are shown in Table 56.

[Test Example 14: Effect of Each Substrate on MDCK Cell Proliferation Action]
Cellulose nanofibers (PNC), chitin nanofibers (biomass nanofibers BiNFi-S (Binfis) 2% by mass, Sugino Machine Co., Ltd.) and deacylated gellan gum (KELCOGEL CG-LA, tricrystals) prepared in Production Example 2 Solution) in 1% (w / v) ultrapure water (Milli-Q water) and then dissolved by stirring while heating at 90 ° C., and this aqueous solution is dissolved at 121 ° C. for 20 minutes. Autoclave sterilization. Serum-free medium KBM220 medium (made by Kojin Bio Co., Ltd.) A medium composition in which 0.01% (w / v), 0.03%, 0.1% cellulose nanofibers are added to a final concentration, Serum-free medium KBM220 medium Final concentration 0.01% (w / v), 0.03%, medium composition supplemented with 0.1% chitin nanofibers, serum-free medium KBM 220 medium (Cordin Bio) final concentration 0.005% Preparation of (w / v), 0.015%, 0.03%, 0.06%, 0.1% deacylated gellan gum-added medium composition, and non-supplemented medium composition not containing the above base material did. Subsequently, the canine renal tubular epithelial cell line MDCK (DS Pharma Biomedical Co., Ltd.) cultured overnight on the medium from which the serum has been removed (stavation treatment) is added to each of the above-mentioned base materials to 100 000 cells / mL. After seeding with the added medium composition, 100 μL per well was distributed to the wells of a 96-well flat bottom ultra-low adhesion surface microplate (Corning # 3474). Each plate was cultured in a stationary state in a CO ₂ incubator (37 ° C., 5% CO ₂ ) and continued for 14 days. 3, 7, 10, 14 days of ATP reagent ^{100μL (CellTiter-Glo TM Luminescent Cell} Viability Assay, Promega Corp.) with respect to culture solution was added to suspend the, was allowed to stand at room temperature for about 10 minutes, FlexStation3 The luminescence intensity (RLU value) was measured by (Molecular Devices, Inc.), and the number of living cells was determined by subtracting the luminescence value of the medium alone.

  As a result, when MDCK cells are cultured on a low adhesion plate using deacylated gellan gum, nanocellulose fiber PNC, and chitin nanofibers which are the medium composition of the present invention, the growth promotion of MDCK cells with all substrate additions An action was observed. Among them, chitin nanofibers showed the strongest effect. The RLU values (ATP measurement, luminescence intensity) in each culture are shown in Table 57.

[Test Example 15: Effect of Chitin Nanofiber on MDCK Proliferation Action]
Cellulose nanofibers (PNC), chitin nanofibers (biomass nanofibers BiNFi-S (Binfis) 2% by mass, Sugino Machine Co., Ltd.) and deacylated gellan gum (KELCOGEL CG-LA, tricrystals) prepared in Preparation Example 1 Solution) in 1% (w / v) ultrapure water (Milli-Q water) and then dissolved by stirring while heating at 90 ° C., and this aqueous solution is dissolved at 121 ° C. for 20 minutes. Autoclave sterilization. Serum-free medium KBM220 medium to a final concentration of 0.0001% (w / v), 0.0003%, 0.001%, 0.003%, 0.01%, 0.02%, 0.03% chitin nano Medium composition supplemented with fiber, medium composition supplemented with serum-free medium KBM 220 medium (Cordin Bio) with a final concentration of 0.005% (w / v), 0.015%, 0.03% deacylated gellan gum An additive medium composition was prepared, which did not contain the above-mentioned substrate and the above-mentioned substrate. Subsequently, the canine kidney tubular epithelial cell line MDCK (DS Pharma Biomedical Co., Ltd.) cultured overnight on the medium from which the serum has been removed (stavation treatment) is deacylated gellan gum as described above to have 100,000 cells / mL or After seeding with the medium composition to which chitin nanofibers were added, 100 μL per well was dispensed to the wells of a 96-well flat bottom ultra-low adhesion surface microplate (Corning # 3474). Each plate was cultured in a stationary state in a CO ₂ incubator (37 ° C., 5% CO ₂ ) and continued for 14 days. 5, 9, 12, 15 days of ATP reagent ^{100μL (CellTiter-Glo TM Luminescent Cell} Viability Assay, Promega Corp.) with respect to culture solution was added to suspend the, was allowed to stand at room temperature for about 10 minutes, FlexStation3 The luminescence intensity (RLU value) was measured by (Molecular Devices, Inc.), the luminescence value of the medium alone was subtracted, and the number of living cells was measured as an average value of 3 points.

  As a result, when MDCK cells were cultured on a low adhesion plate using the deacylated gellan gum and chitin nanofibers which are the medium composition of the present invention, the growth promoting effect of MDCK cells was observed with the addition of both substrates. Among them, chitin nanofibers showed a growth promoting effect at a concentration of 0.0001% or more, and in particular, showed a high effect at 0.001% or more. The RLU values (ATP measurement, luminescence intensity) in each culture are shown in Table 58.

[Test Example 16: The effect of chitin nanofibers on the proliferation of MDCK cells]
Initial culture;
Ultrapure water so that cellulose nanofibers (PNC) and chitin nanofibers (biomass nanofibers BiNFi-S (Binfis) 2% by mass, Sugino Machine Co., Ltd.) prepared in Production Example 2 become 1% (w / v) After suspending in (Milli-Q water), it was dissolved by stirring with heating at 90 ° C., and this aqueous solution was autoclaved at 121 ° C. for 20 minutes. Serum-free medium KBM220 medium to which a final concentration of 0.01% (w / v) of chitin nanofibers is added, serum-free medium KBM220 medium (Cordin Bio) and chitin nanofiber-free medium composition Prepared. Subsequently, the canine renal tubular epithelial cell line MDCK (DS Pharma Biomedical Co., Ltd.) cultured overnight (in starvation treatment) in a serum-depleted medium is added with the above-mentioned chitin nanofibers to 75,000 cells / mL. After seeding with the above medium composition, the mixture was dispensed to a conical flask 125 ml (Corning # 431405) to a volume of 30 mL per flask. The flask was cultured in a static state in a CO ₂ incubator (37 ° C., 5% CO ₂ ) and continued for 6 days. 0, day 6 dispensed three minutes to 100μL after the culture was suspended in a pipette, ATP reagent 100μL each ^{(CellTiter-Glo TM Luminescent Cell Viability} Assay, Promega Corp.) was added to suspend the approximately After standing at room temperature for 10 minutes, the luminescence intensity (RLU value) was measured with FlexStation 3 (manufactured by Molecular Devices), and the number of living cells was measured by subtracting the luminescence setting of the medium alone.

Subculture;
In order to confirm the effect on subculture, MDCK cells were examined using a cell suspension cultured for 6 days in a medium containing 0.01% chitin nanofibers. Cell suspension prepared by mixing 3 ml of cell suspension and 27 ml of non-supplemented medium composition to make chitin nanofiber concentration 0.001%, 3 ml of cell suspension and final concentration 0.01% (w / v) The cell suspension in which 27 ml of the medium composition to which chitin nanofibers had been added was mixed to make the chitin nanofiber concentration 0.01% was dispensed to each 125 ml Erlenmeyer flask. The flask was cultured in a static state in a CO ₂ incubator (37 ° C., 5% CO ₂ ) and continued for 14 days. 0, 7, 14 days aliquoted three points 100μL after the culture was suspended in a pipette min, was added suspended ATP reagent 100μL each ^{(CellTiter-Glo TM Luminescent Cell Viability} Assay, Promega Corp.) After leaving for 10 minutes at room temperature, measure the luminescence intensity (RLU value) with FlexStation 3 (manufactured by Molecular Devices), subtract the luminescence value of the medium alone, and measure the number of viable cells as an average of 3 points did.

  As a result, when MDCK cells were cultured on an Erlenmeyer flask using chitin nanofibers, which is the medium composition of the present invention, a growth promoting effect of MDCK cells was observed. Furthermore, when the medium containing chitin nanofibers was supplemented, the growth of MDCK cells was observed, and it became clear that the subculture could be carried out easily without treatment with trypsin or the like. The RLU values (ATP measurement, luminescence intensity) in the initial culture are shown in Table 59, and the RLU values (ATP measurement, luminescence intensity) in the subculture are shown in Table 60.

[Test Example 17: Comparison of the growth promoting effect of chitin nanofibers in MDCK cells in each medium]
Cellulose nanofibers (PNC), chitin nanofibers (biomass nanofibers BiNFi-S (Binfis) 2% by mass, Sugino Machine Co., Ltd.) and deacylated gellan gum (KELCOGEL CG-LA, tricrystals) prepared in Preparation Example 1 Solution) in 1% (w / v) ultrapure water (Milli-Q water) and then dissolved by stirring while heating at 90 ° C., and this aqueous solution is dissolved at 121 ° C. for 20 minutes. Autoclave sterilization. Medium composition obtained by adding 0.01% (w / v) of chitin nanofibers to a final concentration of 0.001% (w / v) to serum-free medium KBM220 medium (Cordin Bio) or Cosmedium 012 medium (Cosmo Bio) A medium composition prepared by adding a final concentration of 0.015% (w / v), 0.03% of deacylated gellan gum to serum medium KBM220 medium or Cosmedium 012 medium, and a non-added medium composition not containing the above base material are prepared. did. Subsequently, the canine kidney tubular epithelial cell line MDCK (DS Pharma Biomedical Co., Ltd.) cultured overnight on the medium from which the serum has been removed (stavation treatment) is deacylated gellan gum as described above to have 100,000 cells / mL or After seeding with the medium composition to which chitin nanofibers were added, 100 μL per well was dispensed to the wells of a 96-well flat bottom ultra-low adhesion surface microplate (Corning # 3474). Each plate was cultured in a static state in a CO ₂ incubator (37 ° C., 5% CO ₂ ) and continued for 12 days. 4, 8, 12 day ATP reagent ^{100μL (CellTiter-Glo TM Luminescent Cell} Viability Assay, Promega Corp.) with respect to culture solution was added to suspend the, was allowed to stand at room temperature for about 10 minutes, FlexStation3 (Molecular The luminescence intensity (RLU value) was measured by Devices), and the luminescence value of the medium alone was subtracted, and the number of living cells was measured as an average value of 3 points.

  As a result, when MDCK cells were cultured on a low adhesion plate using the deacylated gellan gum and chitin nanofibers which are the medium composition of the present invention, the growth promoting effect of MDCK cells was observed with the addition of both substrates. Among them, chitin nanofibers showed high growth ability even in the condition using Cosmedium 012 medium at a concentration of 0.001% or more. Under microscopic observation of the cell condition on the 4th day, the medium condition using deacylated gellan gum only disperses the cell clumps (spheres), but the medium condition using chitin nanofibers makes the sphere and cells Was observed to grow like a bunch of grapes. The RLU values (ATP measurement, luminescence intensity) in the culture using the KBM220 medium are shown in Table 61, and the RLU values (ATP measurement, luminescence intensity) in the culture using the Cosmedium 012 medium are shown in Table 62. The results of microscopic observation of the 4-day culture are shown in FIG.

[Test Example 18: Comparison of MDCK cell proliferation action of chitosan nanofibers and chitin nanofibers]
Chitosan nanofibers (biomass nanofibers BiNFi-S, 1% by mass, Sugino Machine Co., Ltd.) and chitin nanofibers (biomass nanofibers BiNFi-S (Binfis) 2% by mass, Sugino Machine Co., Ltd.) and the same as in Reference Example 1 And deacylated gellan gum (KELCOGEL CG-LA, manufactured by Sanyo Co., Ltd.) to 1% (w / v) in ultra pure water (Milli-Q water) and then stirred at 90 ° C. The aqueous solutions prepared were autoclaved at 121 ° C. for 20 minutes. Serum-free medium KBM220 medium (made by Kojin Bio Co., Ltd.) A medium obtained by adding 0.001% (w / v), 0.003%, 0.01% or 0.03% of chitosan nanofibrous or chitin nanofibers to a final concentration Composition, Medium composition prepared by adding serum-free medium KBM220 medium to a final concentration of 0.015% (w / v), 0.03% deacylated gellan gum, and non-supplemented medium composition not containing the above substrate did. Subsequently, the canine renal tubular epithelial cell line MDCK (DS Pharma Biomedical Co., Ltd.) which has been cultured overnight in a medium from which serum has been removed (stavation treatment) is deacylated gellan gum as described above to have 100,000 cells / mL. After seeding with a medium composition to which chitosan nanofibers or chitin nanofibers were added, 100 μL per well was dispensed to the wells of a 96-well flat bottom ultra-low adhesion surface microplate (Corning # 3474). Each plate was cultured in a static state in a CO ₂ incubator (37 ° C., 5% CO ₂ ) and continued for 12 days. 7, day 11 of ATP reagent ^{100μL (CellTiter-Glo TM Luminescent Cell} Viability Assay, Promega Corp.) with respect to culture solution was added to suspend the, was allowed to stand at room temperature for about 10 minutes, FlexStation3 (Molecular Devices, Inc. Luminous intensity (RLU value) was measured by the product, and the number of living cells was measured as the average value of 3 points by subtracting the luminescence value of the medium alone.

  As a result, when MDCK cells were cultured on a low adhesion plate using chitosan nanofibers and chitin nanofibers which are the medium composition of the present invention, a growth promoting effect higher than that of deacylated gellan gum was observed. In addition, chitin nanofibers showed high proliferation ability even under the condition of 0.001% concentration, but chitosan nanofiber showed high proliferation ability from the concentration of 0.01%. The RLU values (ATP measurement, luminescence intensity) are shown in Table 63.

[Test Example 19: Fresh cynomolgus monkey primary hepatocyte preservation test]
Cellulose nanofibers (PNC) prepared in Preparation Example 2 and 1% by mass (w / v) aqueous solution of κ-carrageenan (GENUGEL WR-80-J, manufactured by Three Crystal Co., Ltd., in the same manner as in Example 5) Made and used. A medium composition prepared by adding a final concentration of 0.03% (w / v), 0.1% PNC or carrageenan to Williams' E medium (manufactured by Life Technology Co., Ltd.) containing 10% FBS, and not containing the above substrate A supplemented media composition was prepared. Subsequently, fresh cynomolgus monkey primary hepatocytes (manufactured by Ina Research Co., Ltd.) are mixed with the above-mentioned medium composition to which PNC or carrageenan is added so as to be 2,500,000 cells / mL, and then Cryogenic vials for cell freezing. It dispensed to (made by Thermo Scientific company). In addition, the thing which suspended cynomolgus monkey primary hepatocytes in the same culture medium which does not contain a base material was dispensed. The above operation was carried out in 2 lots. Subsequently, the tube was transported still for 2 days under refrigeration (about 4 ° C.). After transportation under refrigeration conditions for 2 days, cell viability in the suspension was measured using Trypan Blue reagent (manufactured by Life Technology) on the cell suspension.

  As a result, when fresh monkey primary hepatocytes were transported under refrigeration using PNC which is the medium composition of the present invention, the survival rate was higher than that in the non-added condition. On the other hand, carrageenan did not show such an effect. The survival rates are shown in Table 10.

[Test Example 20: Evaluation of proliferation after reseeding]
The mouse preadipocyte cell line 3T3-L1 (manufactured by ATCC) was seeded on a 10 cm polystyrene Petri dish using DMEM medium containing 10% FBS, and cultured in an incubator set at 37 ° C., 5% CO ₂ . After the 3T3-L1 cells become confluent, the medium is aspirated and removed, and the FBS is removed with D-PBS (manufactured by Wako Pure Chemical Industries, Ltd.), 1 ml of a solution containing 0.25% Trypsin and 1 mM EDTA (manufactured by Wako Pure Chemical Industries, Ltd.) Was added to the above polystyrene petri dish. After confirmation of cell detachment, 10% by volume of FBS-containing DMEM medium was added to recover the cells from the petri dish and transferred to a centrifuge tube. After centrifugation at 300 × g, the supernatant was removed. Perform 150 μl of cell suspension added to a 1.5 mL microtube as a cell suspension of approximately 200 × 10 ⁴ cells / mL, and prepared in advance to contain 10% (v / v) FBS The medium composition of Example 2 (PNC concentration 0.06%) to Example 4 (DAG concentration 0.03%), Comparative Example 5 (Alg concentration 0.03%) and DMEM medium containing 10% by volume FBS as a negative control 150 μL of each was added, and cell suspension (about 100 × 10 ⁴ cells / mL) was prepared by pipetting.

After storing in a sealed state for 7 days under stationary conditions at room temperature, a portion of the cell suspension is diluted with DMEM medium containing 10% FBS, and approximately 10 × 10 ^{4 based on} the seeding concentration before storage for 7 days. A cell suspension of cells / mL was prepared. 96 well multi-plates seeded with cell suspensions by 100μL to (Corning), seeded the day, with the addition of ATP reagents 100μL later and after 2 days 1 day ^{(CellTiter-Glo TM Luminescent Cell Viability} Assay, Promega Corp.) After standing at room temperature for 15 minutes, the luminescence intensity (RLU value) was measured (n = 5) with FlexStation 3 (manufactured by Molecular Devices), and the number of living cells was measured by subtracting the luminescence value of the medium alone.

  As a result, the number of viable cells (RLU value) of the medium composition of the negative control or Comparative Example 5 on the day of reseeding after storage for 7 days is significantly lower than the medium compositions of Examples 2 to 4. It became. The number of viable cells (RLU value) one day after re-seeding was increased as compared with the day of re-seeding on each of Example 2 and Example 4, and the cells after storage also retained proliferative properties. The results for the number of viable cells are shown in Table 65.

  The medium composition according to the present invention exhibits an excellent cell and / or tissue floating effect, and is extremely useful in culturing a large number of cells and / or tissues derived from animals and plants while maintaining their functions. In addition, cells and / or tissues cultured by the method of the present invention are lost due to evaluation of efficacy and toxicity of chemicals, pharmaceuticals and the like, large-scale production of useful substances such as enzymes, cell growth factors and antibodies, and diseases and defects. It is extremely useful in the field of regenerative medicine that supplements the organs, tissues and cells.

  The contents described in all the publications, including the patents and patent applications mentioned herein, are hereby incorporated by reference in their entirety to the same extent as if expressly set forth. .

  This application is related to Japanese Patent Application No. 2014-010842 (filing date: January 23, 2014) filed in Japan, Japanese Patent Application No. 2014-123772 (filing date: June 16, 2014), Japanese Patent Application No. 2014-174574 (filing date) No. 2014-217761 (filing date: October 24, 2014), the contents of which are incorporated in full herein.

Claims (4)

Chitin nanofibers in a medium compositions containing in a dispersed state in a liquid medium, comprising suspension culture adherent cells in a state of adhering to the chitin nanofibers, a method of proliferating adherent cells. The method according to claim 1 , wherein the content of chitin nanofibers in the medium composition is not less than 0.0001% (w / v) and not more than 0.1% (w / v). The average fiber diameter of chitin nanofibers is 0.001 to 1.00 μm, and the ratio (L / D) of the average fiber length (L) to the average fiber diameter (D) is 2 to 500. The method described in. The method according to any one of claims 1 to 3, wherein the viscosity of the medium composition is 8 mPa · s or less.